JAAJournal of Amino Acids2090-0112SAGE-Hindawi Access to Research35253810.4061/2011/352538352538Review ArticleThe Catalytic Machinery of a Key Enzyme in Amino Acid BiosynthesisViolaRonald E.1FaehnleChristopher R.2BlancoJulio1MooreRoger A.3LiuXuying4Arachea Buenafe T.1PavlovskyAlexander G.1AhmadShandar1Department of ChemistryUniversity of ToledoToledo, OH 43606USAutoledo.edu2Structural Biology LabCold Spring Harbor LabsCold Spring HarborNY 11724USAcshl.edu3Rocky Mountain LabsNational Institutes of HealthHamilton, MT 59840USAnih.gov4Department of PharmacologyYale UniversityNew Haven, CT 06520USAyale.edu201125112010201102102010071120102011Copyright © 2011 Ronald E. Viola et al.This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

1. Introduction

Enzyme-catalyzed reactions organized into sequential pathways are responsible for producing the molecules needed to sustain life. While many essential metabolic pathways are present in all known life forms, there are also significant differences between microbial and mammalian metabolism. Most microbial species can synthesize all of their important metabolic building block molecules, while mammals must acquire many of these from dietary sources. These metabolic differences provide a substantial number of potential protein targets to be examined for the development of selective antimicrobial agents. There is a growing need to identify effective new antibiotics that function against new targets to combat the expanding threat from pathogenic species that are becoming increasingly resistant to existing antibiotics [1, 2]. There are, however, fundamental problems that must be resolved for effective biocides to be developed against metabolic enzyme targets. Infectious microorganisms often use their host as a source of essential metabolites, thus bypassing inhibitors designed to block key steps in microbial metabolism. In many instances microorganisms have developed alternative routes for the production of important metabolites that can readily bypass inhibited enzyme reactions [3]. Essential enzymatic pathways can also be altered in response to an antibiotic threat. Although these microbial pathways may not exist in mammals, related enzymes with similar active site geometries or substrate binding motifs can potentially interact with even the most carefully designed inhibitors. The structure and mechanism of a bacterial metabolic enzyme must be thoroughly characterized before a potential drug target can be fully evaluated. Aspartate β-semialdehyde dehydrogenase (ASADH) is a key enzyme in an essential amino acid biosynthetic pathway that is not present in mammals. This enzyme has been thoroughly investigated and is now being examined as a target for the development of new antimicrobial agents.

2. The Aspartate Biosynthetic Pathway in Microbes

The aspartate biosynthetic pathway is present only in plants and microbes. The commitment step to this pathway is the phosphorylation of aspartic acid catalyzed by a family of aspartokinases (AK). The next enzyme in the pathway, ASADH, catalyzes the production of aspartate semialdehyde (ASA) that is located at a critical junction in this pathway. From this point one pathway branch leads to the production of lysine through the metabolite diaminopimelate, while the alternative route leads to the synthesis of methionine, threonine, and isoleucine with homoserine serving as their common intermediate (Figure 1). Thus, one quarter of the amino acids required for protein synthesis in all organisms are linked through and synthesized by this pathway [4]. The aspartate pathway is exquisitely regulated to control the total amino acid output as well as the relative levels of each amino acid. This regulatory scheme must also maintain the levels of several essential metabolic intermediates during periods of low protein synthesis. This is accomplished by coordinated regulation by the end product amino acids, both through feedback inhibition and by selective gene repression [5]. This regulatory scheme allows each end product to modulate the flux through the initial pathway steps, with branch point allosteric enzymes providing further control over the product levels.

The aspartate biosynthetic pathway in microorganisms. The end product amino acids produced by this pathway are shown in green. Pathway-specific metabolites (shown in red) play crucial roles in microbial life cycle functions (shown in blue).

In addition to the essential amino acids produced in the aspartate pathway, several important metabolites are synthesized that play crucial roles in important developmental processes, such as cell wall biosynthesis, protective dormancy, and virulence factor production. For example, dihydrodipicolinate is a precursor of dipicolinate, the major component of bacterial spores [6], and diaminopimelate (DAP) is required for cross-linking of the peptidoglycan polymers [7] in bacterial cell wall synthesis (Figure 1). Another product of this pathway, S-adenosylmethionine, is an essential methyl group donor that also serves as a precursor for quorum sensing signaling molecules with critical roles in triggering virulence factors in infectious microorganisms [8, 9]. Homoserine lactone signals, for example, are essential for both virulence and biofilm development in the human pathogen Acinetobacter baumannii [10].

Because this pathway produces many essential compounds that are involved in a range of critical functions, disruptions to the aspartate pathway are fatal to those microorganisms. In particular, selective perturbations of the asd gene that encodes for ASADH are lethal to numerous infectious microorganisms. For example, asd mutants of Salmonella typhimurium develop an absolute growth requirement for diaminopimelate (DAP), a critical cell wall cross-linking component (Figure 1) in Gram-negative bacteria [11]. This mutated organism undergoes cell lysis when DAP is not supplied, and, since this metabolite is not produced in mammals it cannot be supplied by the host organism. A similar loss of viability is observed in asd-deficient E. coli strains. During amino acid starvation microorganisms often use specific transport systems to import exogenous amino acids available from the host environment [12]. However, de novo biosynthesis of lysine is essential for the survival of Mycobacterium tuberculosis during infection in mice, despite the presence of lysine in the host [13]. Even if an organism could mutate to improve lysine transport capacity in response to aspartate pathway inhibition, reversal of the decarboxylation that produces lysine from DAP is neither kinetically nor thermodynamically feasible. Both of these end products and several additional intermediates of this pathway are thus critical for microbial cell viability, both in culture and during host infection. Blockage of the aspartate pathway is fatal to microorganisms. Therefore the identification of effective inhibitors of key aspartate pathway enzymes should provide lead compounds for the development of new biocides. To achieve this aim we have focused on the functional and structural characterization of the microbial ASADH family of enzymes.

3. Sequence and Structural Comparisons among the Aspartate-<bold><italic>β</italic></bold>-Semialdehyde Dehydrogenases

Aspartate-β-semialdehyde dehydrogenase (ASADH) catalyzes the second reaction in the aspartate pathway, the reductive dephosphorylation of β-aspartyl phosphate to aspartate-β-semialdehyde (ASA) (Scheme 1), at a critical branch point in this pathway.

The conversion of aspartate β-semialdehyde to β-aspartyl phosphate catalyzed by aspartate β-semialdehyde dehydrogenase in the reverse (nonphysiological) reaction.

The ASADHs from a variety of organisms encompass a considerable diversity of sequence homologies, ranging from less than 10% to as high as 95% sequence identity when compared to the Escherichia coli enzyme (ecASADH). The ASADH enzymes in microorganisms can be divided into three branches consisting of the enzymes from Gram-negative bacteria, Gram-positive bacteria, and archaea/fungi. These branches were initially identified and partitioned through sequence alignments and now, with representative high resolution structures available from each branch, have been compared by structural alignments. The earliest structures of ASADHs are from enzymes that were isolated and purified from Gram-negative bacteria. These enzyme forms share significant sequence and structural homology and include structures of the ASADHs from E. coli [14, 15], Vibrio cholerae [16], and Haemophilus influenzae [17]. The overall structure of these ASADHs is a homodimer with an extensive contact surface between the subunits. Each monomer is composed of a carboxy-terminal domain primarily involved in hydrophobic intersubunit contacts, and a more hydrophilic amino-terminal domain that forms the active site and NADP binding site (Figure 2).

The ASADH from Streptococcus pneumoniae (spASADH) is the first member of the Gram-positive bacterial branch that was structurally characterized [18]. spASADH is a good representative of the other Gram-positive bacterial ASADHs with greater than 40% sequence identity to these enzymes, while having less than 25% identity with any of the Gram-negative enzymes. Unexpectedly, this spASADH has lower sequence homology to the Gram-negative enzymes than to those of the archaeal/fungal branch, with sequence identities from this comparison ranging from 18 to 30%. The structure of the ASADH from the archaeal hyperthermophile Methanococcus jannaschii (mjASADH) has a similar overall fold and domain arrangement as the Gram-negative enzyme forms [19] despite being less than 10% sequence identity. But the complete set of functionally important active site amino acids has been conserved in mjASADH, suggesting an identical mechanism in the enzyme from this ancient organism despite its lower catalytic efficiency. The first structure of an ASADH from a fungal species, Candida albicans (caASADH), was recently determined, and it also possesses a similar overall fold, domain organization, and active site structure as the other members of this enzyme family [20]. This fungal enzyme has less than 30% sequence identity to any of the bacterial ASADHs and only 40% identity to its closest homologue. Alignment of the structures that have been determined from each branch of the ASADH family shows that both spASADH and caASADH are most similar to the archaeal ASADH from M. jannaschii and more distantly related to the Gram-negative enzymes.

Despite the overall sequence and structural similarities, there are a number of insertions and deletions which serve to differentiate the three ASADH branches. Structural variations between these branches include a sequence of residues on the enzyme surface that contribute to the binding pocket for the coenzyme NADP. The E. coli ASADH is representative of the Gram-negative enzymes, with a highly flexible coenzyme binding loop in the absence of NADP that becomes ordered in response to NADP binding [15]. The archaeal mjASADH has three conserved insertions totalling 30 residues when aligned against the Gram-negative bacterial ecASADH [19], with each insertion located on the surface of the structure. A 13-residue insertion in mjASADH leads to an alternative orientation of the coenzyme binding loop, differing from that in the Gram-negative enzymes by about 90°, causing it to drape over and occlude the coenzyme binding pocket [19]. In addition, the helical subdomain that comprises a significant fraction of the dimer interface (Figure 2) is absent in the archaeal mjASADH. The Gram-positive spASADH has the same number of residues in this region as the Gram-negative bacterial enzymes, but two short β-strands replace the helix-turn-helix structure observed in the helical subdomain of ecASADH (Figure 2). The fungal enzyme from C. albicans is also missing the helical subdomain [20] and contains most of the insertions and deletions observed in the archaeal enzyme. These structural changes suggest differences in how each branch of this enzyme family can carry out its catalytic role, even though each possesses an identical repertoire of highly conserved active site functional groups.

4. Role of Active Site Functional Groups

In spite of the overall sequence diversity between the different branches of the ASADH family the identity of the core active site functional groups has been preserved throughout evolution (Figure 3). A set of active site mutants of ASADH from H. influenzae (hiASADH) was examined kinetically and structurally with the goal of more precisely establishing the role for each functional group in substrate recognition and binding. A cysteine had been previously identified from biochemical studies as the likely active site nucleophile [21]. Replacement of a single sulfur atom with an oxygen in the C136S mutant virtually eliminates catalysis (Table 1), supporting the essential role of this residue as the catalytic nucleophile. Some decrease in catalytic activity would be expected because the hydroxyl group of serine is a weaker nucleophile than a cysteine sulfhydryl group. Only minor changes are observed in the active site structure of this mutant despite the nearly complete loss of catalytic activity. But a change in orientation of the introduced hydroxyl group is likely responsible for the very low activity of this mutant. The side chain hydroxyl group of the introduced serine rotates by about 90 degrees, with this new orientation stabilized by a hydrogen-bond to an adjacent backbone carbonyl group [22]. This reorientation not only moves this functional group out of the position needed to act as a nucleophile, but also competes with the involvement of this backbone carbonyl in intermediate stabilization and alters the position of the bound phosphate group.

Kinetic and structural consequences of ASA dehydrogenase active site mutants.

Active site groupaProposed functionMutantCatalytic activitybStructural effect of the mutationPDB code
Cys136 (Cys134)Active site nucleophileC136S<0.01%Rotation of S136 shifts the amino group of ASA and moves the bound phosphate by 1 Å1PQP

His277 (His274)Acid-base catalystH277N1.0%Breakdown of the initial tetrahedral intermediate is slowed by a shift in the phosphate position1PQU

Arg270 (Arg267)Binds the substrate carboxyl groupR270K R267L0.1%9.5%The introduced lysine moves about 2 Å away from the original arginine position1PS8

Glu243 (Glu240)Binds the substrate amino groupE243D1.2%The bound intermediate shifts position by about 0.5 Å towards the shorter side chain of D2431Q2X

Lys246Helps orient bound phosphateK246R3.3%The introduced arginine rotates by about 90° to form new interactions with S99 and K2421PU2

Arg103Binds phosphateR103K R103L0.4%0.07%Displaces the active site loop (N135 to S139) which shifts C136 away from H2771PR3 1OZA

aH. influenzae sequence numbering, with the numbers in parenthesis referring to the V. cholerae sequence

bkcat determined by varying ASA at fixed NADP levels and expressed as a percent of wild type enzyme activity

The active site structure of Vibrio cholerae ASADH with bound NADP and the covalent inactivator SMCS (shown in green). Cys134 is the active site nucleophile, and His274 is the acid-base catalyst. Glu240 and Arg267 are substrate binding groups, with Arg101 and Lys243 comprising part of the phosphate binding site that is occupied in this structure by a water molecule (W2). H. influenzae numbering is in parentheses (figure adapted from reference [16]).

Mammalian glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes a similar reaction, an oxidative phosphorylation, to the reverse reaction catalyzed by ASADH and appears to do so by the same mechanism [23]. Before the first structure of ASADH was available, sequence alignments to the mechanistically related enzyme (GAPDH) family were used to identify and then test the roles of possible active site functional groups. Both enzymes were proposed to contain a cysteine-histidine catalytic dyad; however the corresponding position of the catalytic histidine in GAPDH is occupied by a conserved glutamine in ASADH [24]. Replacement of this functional amino acid causes a loss in catalytic activity that is not fully recovered even when a histidine is introduced at this position in ASADH. This mystery was solved when the first ASADH structure revealed that the essential histidine residue actually comes from a completely different position in the primary sequence, with a loop containing H277 folded into the enzyme active site [14]. The H277N mutant of hiASADH has significantly impaired activity (Table 1), highlighting the key role for this residue. However a ternary complex structure with NADP and the active site-directed inactivator, S-methyl-L-cysteine sulfoxide (SMCS), showed continuous density extending from the Cys136 nucleophile [22]. This structure is consistent with the covalent attachment of SMCS to the active site nucleophile. The position of this bound inactivator is shifted in the H277N mutant relative to its position when bound in the wild-type enzyme [15]. This reorientation moves this compound further from the bound phosphate that must attack the carbonyl carbon of the acyl-enzyme intermediate to generate β-aspartyl phosphate in the reverse reaction (Scheme 1).

So in summary for these catalytic mutants, the C136S mutant still allows substrate binding but formation of the covalently bound intermediate is slowed by a less efficient and misoriented nucleophile. In contrast, the H277N mutant appears to form the initial tetrahedral intermediate efficiently, but its subsequent breakdown is hindered by a shift away from the bound phosphate nucleophile. In each case, small perturbations in the positioning of essential catalytic groups or reactive intermediates are shown to have dramatic effects on enzyme catalysis.

Additional mutant structures of hiASADH have been determined, with each substitution replacing a putative substrate binding group in order to assess their function. Each of these mutants displayed significantly impaired catalytic activity, ranging from ~10% to less than 0.1% that of the wild-type enzyme [25]. However, the structural basis for the diminished activities is different for each mutation (Table 1). A conserved arginine (Arg270) in the ASADH family aligns with a conserved Arg231 in the GAPDH family, a residue that had previously been assigned a role in binding the substrate phosphate group [26]. Based on kinetic studies this arginine residue was proposed to have a comparable role in hiASADH, namely, binding the carboxyl group of the substrate ASA [24]. The structures of substrate complexes of the wild-type enzyme [17] and of the inactivator SMCS covalently bound to Vibrio cholerae ASADH (vcASADH) [16] support this assignment by clearly establishing the presence of a bidentate interaction between Arg270 and the carboxylic group of the substrate ASA.

Arginine270 was replaced by lysine in hiASADH to assess its relative importance in substrate binding by disrupting the bidentate interaction that is formed with ASA. Maintaining this interaction between the introduced lysyl amino group and the ASA carboxyl group will require a shift in the orientation of both the lysyl side chain and the resulting tetrahedral intermediate relative to their positions in the wild-type enzyme. The dramatic loss of activity in R270K can be explained by a shift of the intermediate away from the position that is necessary to form and maintain a productive interaction between its carboxyl group and the altered substrate binding residue during the catalytic cycle. A neutral side chain introduced at this position in ecASADH (R267L) cannot form any binding interaction with the substrate carboxyl group, leading to a 30-fold increase in the Km for ASA [24] while also permitting greater flexibility for positioning of the covalent intermediate. This flexibility increases the likelihood that the intermediate can adopt a catalytically viable conformation relative to that imposed in the hiASADH R270K mutant, and this conformational flexibility is manifest in the 100-fold greater activity seen in the R267L ecASADH enzyme form (Table 1).

Glutamate243 provides a side chain carboxyl group in the active site of ASADH whose role in the catalytic cycle had not been definitively established. This group is highly conserved among ASADHs from different organisms, and structural studies have shown that it is in position to potentially interact with the amino group of the tetrahedral intermediate in the hiASADH complex [17]. An E243D mutant was produced to assess the possible role of this residue in substrate and intermediate binding. Kinetic studies of E243D failed to show the expected detrimental effect on substrate interactions, with the Km for ASA unchanged from that of the wild-type enzyme. Instead the catalytic efficiency of this mutant is significantly compromised, reducing the kcat value to about 1% of that of wild-type hiASADH (Table 1). In this case the position of the bound intermediate (and presumably that of the bound substrate) shifts towards the shorter side chain of the introduced aspartate at this position. This shift allows this mutant to maintain substrate binding affinity, but compromises the positioning between the intermediate and the bound cofactor thereby leading to impaired catalytic efficiency.

Each of these mutations was prepared with the aim of removing and testing critical substrate binding groups. However the structural rearrangements that occur as a consequence of these replacements manifest themselves not in a loss in substrate binding affinity, but in a loss of catalytic activity. Structural characterization of these mutants shows that only subtle changes in key active site residues, such as rotation of a side chain to form a new hydrogen-bond or a shift in position relative to a catalytic intermediate, are sufficient to adversely affect catalysis.

The phosphate-binding site of ASADH is capable of accommodating different tetrahedral oxyanion analogues [27], leading to different functional consequences. Both oxyanion substrates and oxyanion inhibitors bind in the same position by using the same set of ligands (Figure 4), raising the question of what distinguishes a substrate from an inhibitor? In the apoenzyme the side chain hydroxyl of Thr137 forms a hydrogen-bond with Asn135, and this pairing prevents any interactions with the active site Glu243. Upon binding of either oxyanion substrate, phosphate or arsenate, Thr137 switches hydrogen-bonding partners through a 60° rotation that disrupts its interaction with Asn135. This new conformation moves the threonine hydroxyl group into position to interact with and orient Glu243, an important substrate binding group. Glu243 remains in this substrate binding position in the arsenate structure, and this hydrogen-bond with the Thr137 hydroxyl group persists even in the absence of bound ASA. So, the presence of an oxyanion substrate in the active site helps to position Glu243 to interact with the substrate. However, in the presence of the inhibitor periodate this threonine does not change position and switch partners. In this inhibitor-bound structure Thr137 remains hydrogen-bonded to Asn135, which is now oriented away from Glu243 and cannot stabilize the position of this side chain. This single change in hydrogen-bonding partners is apparently sufficient to interfere with the binding of ASA. Thus, subtle shifts in the position of side chains, even those not directly involved in substrate binding or catalysis, can lead to a sequence of events that result in loss of function for a finely tuned enzyme catalyst.

The oxyanion binding site in H. influenzae ASADH. Each of the oxyanion substrates and inhibitors interacts with the same protein ligands, Arg103 and Lys246, and is bound within attacking distance of the covalent acyl-enzyme intermediate. In the substrate structures with either phosphate or arsenate a second oxyanion molecule is bound to Ser100, Lys242, and Lys246 (figure adapted from reference [28]).

5. Differences in Coenzyme Binding and Specificity

The active site functional groups of ASADH are already poised to accommodate amino acid substrate binding in the apoenzyme. However, the binding of NADP is required to induce a domain closure that sets up the active site for catalysis. NADP binding and the coupled domain closure are driven by numerous interactions between the enzyme and the molecular features that are distributed throughout the coenzyme. In vcASADH, Arg9 is repositioned during NADP binding to form an electrostatic interaction with the 2′-phosphate, with additional hydrogen-bonds to phosphate from Thr36 and Ser37 (Figure 5). A consensus sequence, SGxG, present in each branch of the ASADH family interacts via backbone carbonyl hydrogen-bonds to the amide nitrogen of the nicotinamide, while a conserved glutamine (Gln350) in the bacterial enzymes and a corresponding asparagine in the archaeal enzyme are in position to hydrogen-bond to the amide oxygen (Figure 5).

Binding interactions to the adenine ring of NADP are less conserved between the Gram-negative and Gram-positive bacterial enzymes, leading to some drastic differences in coenzyme binding. The adenine base in vcASADH is oriented by a cation-π interaction with Arg9 (Figure 5) in the consensus GxxGxxG sequence which is part of the Rossmann nucleotide fold [29]. A surface loop spanning from Leu189 to Ser195 closes around NADP in these Gram-negative enzymes, with the exocyclic N6 of the adenine base forming a hydrogen-bond with the backbone carbonyl of a proline (Pro192) located on the helical subdomain from the opposite subunit of the dimer (Figure 2). This interaction plays a critical role in the change from an open to a closed enzyme conformation upon coenzyme binding [16]. The overall domain movements encountered in the transition from the apoenzyme to the NADP complex in Gram-positive spASADH are similar to those observed for vcASADH [15]. Rotation of the N-terminal domain toward the active site in response to NADP binding appears to be a universal mechanism utilized throughout the ASADH family. However, none of the interactions that drive domain closure in both ecASADH and vcASADH are observed in spASADH. Thus, facilitation of the commonly observed domain closure in spASADH must be driven by a different set of interactions than those that drive the same closure in the Gram-negative ASADHs.

A completely new binding pocket for the adenine base is found in spASADH, along with an altered 2′-phosphate binding site, from that previously observed in the Gram-negative enzymes. The adenine binding pocket in spASADH is forged between an α-helix and the coenzyme binding loop of the N-terminal domain. Interactions between the adenine ring and the side chains of Thr76 and Ser37, along with a cation-π interaction with Arg39, serve to anchor the adenine ring in this pocket [18]. These new interactions cause the adenine ring to adopt an altered position in spASADH formed by rotation around the bonds linking the nicotinamide and adenine ribose with the bridging diphosphates. As a consequence the center of the adenine ring is shifted by about 8.5 Å with respect to its position in NADP bound in vcASADH, and the position of the exocyclic amine on C6 that formerly interacted with Pro192 from the adjacent subunit has been displaced by nearly 14 Å (Figure 5). However, the 2′-phosphate of NADP bound in the spASADH structure moves less than 5 Å relative to its position in vcASADH, and the phosphate binding groups move to accommodate this shift.

Comparison of the coenzyme binding site between archaeal mjASADH and the bacterial enzymes also shows some conserved similarities. In bacterial ASADHs the backbone amino groups from a methionine and valine are hydrogen-bond donors to the pyrophosphate of NADP (Figure 5), and a change from methionine to serine in the mjASADH structure does not alter these pyrophosphate interactions. A similar reorientation of the adenine ring to that observed in spASADH is seen in the binding of NADP to the ASADH from M. jannaschii [19]. An extended surface loop in mjASADH (from residues 40 to 77) forms the binding pocket for this conformation of the bound NADP. Specific contacts between the adenine ring and the enzyme include two hydrophobic interactions to Leu91 and Leu95 that are within 4.0 Å of the plane of the adenine ring and a cation-π interaction with Arg39. Similar interactions and a closely related coenzyme orientation are also found in the binding of the adenine ring in the fungal enzyme structure (caASADH) [20] (Figure 5).

These changes in coenzyme conformation and in the nature of the interactions with the 2′-phosphate of NADP in the mjASADH structure suggest the possibility of altered coenzyme specificity relative to that seen for the well-studied Gram-negative bacterial enzymes. Preference for NADP versus NAD binding in enzymes is mediated through specific interactions with either the 2′-phosphate of NADP or the 2′-hydroxyl of NAD. The ASADHs from E. coli, V. cholerae, and H. influenzae are each highly specific for NADP. The structure of the NADP complex in vcASADH shows that the 2′-phosphate group is bound by the same arginine that stacks against the adenine base, along with three hydrogen bonds that are formed with two serines and a threonine. This arginine in vcASADH is not conserved in mjASADH but instead is replaced by a threonine that is still in the correct position to form a potential hydrogen-bond with the 2′-phosphate of NADP but, unlike in vcASADH, is not capable of binding to the adenine base.

The possibility of expanded coenzyme specificity for the archaeal enzyme was examined kinetically by testing both NADP and NAD as substrates near the physiological temperatures for this thermophilic organism. mjASADH is found to have comparable catalytic rates with either NAD or NADP when examined at 70ºC [19]. However, this enzyme still shows a preference for NADP over NAD as its coenzyme, with a Michaelis constant that is 130-fold lower for NADP (KNADP = 13 μM) compared to that for NAD (KNAD=1700μM). It appears that the relaxed specificity for NADP in mjASADH is primarily due to absence of this critical arginine residue, since the positively charged guanidinium group of this arginine interacts directly with the negatively charged 2′-phosphate of NADP. An additional explanation for this altered coenzyme specificity comes from the structure of the NADP complex in which the adenine and ribose end of the NADP is bound in a completely different binding pocket in archaeal mjASADH compared to the Gram-negative bacterial enzyme family.

6. The Presence of an Intersubunit Communication Channel

The subunit interface in the homodimer for both the bacterial and archaeal ASADHs is composed primarily of hydrophobic β-sheets. The Gram-negative bacterial ASADHs have the largest dimer interface, formed by a conserved hydrophobic β-sheet and complemented by the helical subdomain to create over 3400 Å2 of buried surface area (Figure 2). This subdomain is also present in the Gram-positive bacterial spASADH, but a 16-residue deletion results in a single α-helix followed by an unstructured loop forming the top portion of the dimer interface with about 2600 Å2 of total buried surface area. A 48-amino acid deletion in mjASADH results in the complete removal of the helical subdomain that makes a significant contribution to the dimer interface in ecASADH. As a consequence the thermophilic mjASADH structure has a much smaller dimer interface of about 2000 Å2. The enzyme from C. albicans (caASADH) is also missing the helical subdomain, with the 44 amino acids that constitute this helix-turn-helix motif in the Gram-negative enzyme forms replaced by an unstructured 3 amino acid loop [20]. As a consequence this fungal enzyme has the smallest dimer interface (~1800 Å2) and the lowest percentage of buried surface area among the structurally characterized ASADHs. Interestingly, there is good correlation between the dimer interface area and enzymatic activity, with the enzyme forms possessing the highest buried dimer surface having the highest catalytic activity [30]. This correlation suggests that intersubunit communication in the ASADH dimer is critical to promote highly efficient catalysis.

A network of hydrogen bonds has been identified across the dimerization interface that is proposed to allow active site to active site communication in the functional ASADH dimer from the bacterial enzyme branch [16]. These structural observations support the earlier kinetic experiments that had suggested an alternating site reactivity model for ASADH catalysis [31]. The active site Glu240 in subunit A of vcASADH is positioned through a hydrogen-bond to Gln161 (Figure 3). Gln161 also forms a hydrogen-bond with the backbone amide of Thr159, which in turn is hydrogen-bonded through its side chain hydroxyl group to the hydroxyl group of Tyr160 across the dimer interface in subunit B (Figure 6). A complementary series of interactions links the active site Glu240 in subunit B to Tyr160 in subunit A, such that each active site is linked to an amino acid in the adjacent subunit through a network of three hydrogen-bonds. The orientation of the Tyr160 residues at the domain interface is crucial to maintaining this network. The tyrosine side chain position is stabilized by π-stacking to the tyrosine from the other subunit and also by a perpendicular π-stacking interaction with Phe345 in the same subunit (Figure 6).

Proposed intersubunit communication channel in bacterial ASADHs. The subunit interface of vcASADH (gray) is bridged by a hydrogen-bonding network (dotted lines) connecting the active sites of each subunit and stabilized by π-stacking interactions (dashed lines). In the subunit interface of mjASADH (green) replacement of Tyr160 with Met183 interrupts the hydrogen-bonding network, and replacement of Phe345 with Thr328 disrupts the π-stacking stabilization.

The structure of mjASADH does not allow the intersubunit communication route that is seen in the bacterial enzymes. Two methionines are present within the dimerization interface region of mjASADH that contribute to the hydrophobic subunit interactions, but replace the conserved tyrosines in each subunit that forms the heart of the hydrogen-bonding network bridging between the two active sites (Figure 6). The phenylalanines that stabilize these tyrosines through π-stacking have also been replaced in the archaeal ASADH branch with a conserved threonine (Figure 6). These amino acids that are crucial for intersubunit communication have also been replaced in the caASADH enzyme. The base-stacked tyrosine is substituted by either leucine or methionine at this position in all fungal ASADHs, while the stabilizing phenylalanine is now a conserved valine in this branch of the enzyme family [20]. These amino acid replacements disrupt the intersubunit hydrogen-bonding network observed in Gram-negative ASADHs, leading to the loss of this communication channel. The alternating sites reactivity observed in the bacterial forms of ASADH is likely absent in both the archaeal and fungal enzymes and could explain the very low catalytic activity in these enzyme forms despite the presence of an identical constellation of active site functional groups throughout the ASADH enzyme family.

7. Catalytic Mechanism and Covalent Intermediates

The proposed catalytic mechanism of ASADH in the reverse (nonphysiological) direction involves the initial attack of the active site cysteine nucleophile (Cys136) on the carbonyl carbon of the substrate ASA. The enzyme-bound tetrahedral intermediate produced by this reaction (Figure 7, structure a) is set up to transfer a hydride ion to the NADP coenzyme bound at an adjacent site, leading to an acyl-enzyme intermediate (Figure 7, structure b). Attack of bound phosphate at the carbonyl carbon of this intermediate produces a second enzyme-bound tetrahedral intermediate (Figure 7, structure c). Collapse of this intermediate with expulsion of the enzyme thiolate group yields the β-aspartyl phosphate product and leaves the enzyme ready to bind another molecule of ASA and repeat the catalytic cycle.

Catalytic mechanism of the reversible aspartate β-semialdehyde dehydrogenase-catalyzed conversion of ASA to aspartyl phosphate. (a) Tetrahedral intermediate derived from nucleophilic attack on ASA. (b) Acyl-enzyme intermediate produced by hydride transfer to NADP. (c) Proposed tetrahedral intermediate obtained from phosphate attack on the acyl-enzyme.

The chance to characterize a true intermediate in an enzyme-catalyzed reaction is a rare opportunity to bridge a mechanistic gap that is generally only hypothesized between the substrate and the product complexes, or extrapolated from intermediate-analogue structures. For the ASADHs this feat has been accomplished twice, capturing and structurally characterizing two different reactive intermediates in the catalytic cycle of this enzyme and lending structural support to this proposed mechanism.

A ternary complex produced by diffusing the substrates ASA and phosphate into crystals of hiASADH was examined with the aim of determining the role of different active site groups in the catalytic mechanism. However, what was observed was not the structure of the enzyme-substrate complex, but instead the structure of the actual tetrahedral intermediate in the catalytic cycle of ASADH (structure a in Figure 7). The bound aspartyl intermediate was modeled into the continuous density emanating from the side chain of the cysteine nucleophile [17], and this structure clearly shows the tetrahedral geometry around the carbon that is covalently attached to the active site cysteine thiolate group (Figure 8). Subsequent hydride transfer from this tetrahedral intermediate to NADP would lead to formation of the acyl-enzyme intermediate. However, exclusion of the coenzyme from the crystallization conditions eliminates the hydride transfer step that is required for the reaction to proceed, thus stopping the catalytic cycle at this stage and allowing the determination of this important structure.

The structure of the acyl-enzyme intermediate (structure b in Figure 7) was also determined by running the reaction in the nonphysiological direction. Soaking the spASADH-NADP complex crystals with the substrate ASA allows the catalytic cycle to proceed up to formation of the acyl-enzyme intermediate [18]. Exclusion of phosphate eliminates the nucleophile required to complete the catalytic cycle. Kinetic studies had previously shown that the acyl-enzyme can be quite stable in the absence of phosphate [32]. The refined structures show the key differences between the tetrahedral intermediate and the acyl-enzyme intermediate. The position of the substrate binding groups that interact with these intermediates shift only slightly between these two structures, with the largest movements caused by the conversion from a tetrahedral carbon geometry in the previous intermediate structure to a planar carbon geometry in the acyl-enzyme (Figure 8).

8. Concluding Remarks

Through a combination of kinetic, mutagenic, and structural studies the detailed catalytic mechanism of the aspartate β-semialdehyde dehydrogenases has been defined, and the essential functional groups that play a role in substrate binding, catalysis, and regulation have each been identified. Subtle changes in any of these critical functional groups are sufficient to alter the finely tuned catalytic machinery of this enzyme, leading to enzyme forms with significantly impaired activity. Differences have been observed between the members of this enzyme family, isolated from bacterial, archaeal, and fungal species, regarding their catalytic efficiency, their coenzyme selectivity, and their mode of catalysis. This extensive body of work on the different members of the ASADH family now allows us to evaluate this enzyme as a drug target. Compounds are now being screened [33] that can potentially recognize these structural and functional differences to produce initial inhibitors that can be developed into species-specific antimicrobials against this key enzyme in an essential metabolic pathway.

Aspartate β-semialdehyde

Aspartate β-semialdehyde dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase

S-methyl-L-cysteine sulfoxide

Candida albicans

Escherichia coli

Haemophilus influenzae

Methanococcus jannaschii

Streptococcus pneumoniae

Vibrio cholerae.

This paper is supported by a Grant from the National Institutes of Health (AI077720). Structural studies were conducted at the University of Toledo Macromolecular Crystallography Consortium or at the SBC-CAT, IMCA-CAT, and BioCARS beamlines of the Advanced Photon Source at Argonne National Laboratory. The authors thank the APS staff scientists for their support and assistance with data collection.

MareeC. L.DaumR. S.Boyle-VavraS.MatayoshiK.MillerL. G.Community-associated methicillin-resistant Staphylococcus aureus isolates causing healthcare-associated infectionsEmerging Infectious Diseases20071322362422-s2.0-33846696731ShahN. S.WrightA.BaiG. H.BarreraL.BoulahbalF.Martín-CasabonaN.DrobniewskiF.GilpinC.HavelkováM.LepeR.LumbR.MetchockB.PortaelsF.RodriguesM. F.Rüsch-GerdesS.Van DeunA.VincentV.LasersonK.WellsC.CegielskiJ. P.Worldwide emergence of extensively drug-resistant tuberculosisEmerging Infectious Diseases20071333803872-s2.0-33847414693AlmaasE.OltvaiZ. N.BarabásiA. L.The activity reaction core and plasticity of metabolic networksPLoS Computational Biology2005175575632-s2.0-5544911428710.1371/journal.pcbi.0010068.eorViolaR. E.The central enzymes of the aspartate family of amino acid biosynthesisAccounts of Chemical Research20013453393492-s2.0-003486091710.1021/ar000057qCohenG. N.HerrmannK. M.SomervilleR. L.The common pathway to lysine, methionine, and threonineAmino Acids: Biosynthesis and Genetic Regulation1983Reading, Mass, USAAddison-Wesley147171RagkousiK.EichenbergerP.Van OoijC.SetlowP.Identification of a new gene essential for germination of Bacillus subtilis spores with Ca-dipicolinateJournal of Bacteriology20031857231523292-s2.0-003737873010.1128/JB.185.7.2315-2329.2003Van HeijenoortJ.Recent advances in the formation of the bacterial peptidoglycan monomer unitNatural Product Reports20011855035192-s2.0-003476282110.1039/a804532aLyonG. J.NovickR. P.Peptide signaling in Staphylococcus aureus and other Gram-positive bacteriaPeptides2004259138914032-s2.0-454430568110.1016/j.peptides.2003.11.026ChenX.SchauderS.PotierN.Van DorsselaerA.PelczerI.BasslerB. L.HughsonF. M.Structural identification of a bacterial quorum-sensing signal containing boronNature200241568715455492-s2.0-003720377310.1038/415545aNiuC.ClemmerK. M.BonomoR. A.RatherP. N.Isolation and characterization of an autoinducer synthase from Acinetobacter baumanniiJournal of Bacteriology20081909338633922-s2.0-4254912467810.1128/JB.01929-07GalanJ. E.NakayamaK.CurtissR.Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strainsGene199094129352-s2.0-002511036810.1016/0378-1119(90)90464-3BurkovskiA.KrämerR.Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applicationsApplied Microbiology and Biotechnology20025832652742-s2.0-003618558510.1007/s00253-001-0869-4GokulanK.RuppB.PavelkaM. S.JacobsW. R.SacchettiniJ. C.Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase, an essential enzyme in bacterial lysine biosynthesisJournal of Biological Chemistry20032782018588185962-s2.0-003804319410.1074/jbc.M301549200HadfieldA.KrygerG.OuyangJ.PetskoG. A.RingeD.ViolaR.Structure of aspartate-β-semialdehyde dehydrogenase from Escherichia coli, a key enzyme in the aspartate family of amino acid biosynthesisJournal of Molecular Biology1999289499110022-s2.0-003304700610.1006/jmbi.1999.2828HadfieldA.ShammasC.KrygerG.RingeD.PetskoG. A.OuyangJ.ViolaR. E.Active site analysis of the potential antimicrobial target aspartate semialdehyde dehydrogenaseBiochemistry2001404814475144832-s2.0-003580793710.1021/bi015713oBlancoJ.MooreR. A.KabaleeswaranV.ViolaR. E.A structural basis for the mechanism of aspartate-β-semialdehyde dehydrogenase from Vibrio choleraeProtein Science200312127332-s2.0-003721530810.1110/ps.0230803BlancoJ.MooreR. A.ViolaR. E.Capture of an intermediate in the catalytic cycle of L-aspartate-β -semialdehyde dehydrogenaseProceedings of the National Academy of Sciences of the United States of America20031002212613126172-s2.0-024236317910.1073/pnas.1634958100FaehnleC. R.Le CoqJ.LiuX.ViolaR. E.Examination of key intermediates in the catalytic cycle of aspartate-β-semialdehyde dehydrogenase from a Gram-positive infectious bacteriaJournal of Biological Chemistry20062814131031310402-s2.0-3375005977310.1074/jbc.M605926200FaehnleC. R.OhrenJ. F.ViolaR. E.A new branch in the family: structure of aspartate-β-semialdehyde dehydrogenase from Methanococcus jannaschiiJournal of Molecular Biology20053535105510682-s2.0-2714451879710.1016/j.jmb.2005.09.027AracheaB. T.LiuX.PavlovskyA. G.ViolaR. E.Expansion of the aspartate β-semialdehyde dehydrogenase family: the first structure of a fungal orthologActa Crystallographica D20106622052122-s2.0-7644908813010.1107/S0907444909052834KarstenW. E.ViolaR. E.Identification of an essential cysteine in the reaction catalyzed by aspartate-β-semialdehyde dehydrogenase from Escherichia coliBiochimica et Biophysica Acta199211211-22342382-s2.0-002677050810.1016/0167-4838(92)90360-PBlancoJ.MooreR. A.FaehnleC. R.ViołaR. E.Critical catalytic functional groups in the mechanism of aspartate-beta;-semialdehyde dehydrogenaseActa Crystallographica D20046010180818152-s2.0-1664438978510.1107/S0907444904020104SegalH. L.BoyerP. D.The role of sulfhydryl groups in the activity of D-glyceraldehyde 3-phosphate dehydrogenaseThe Journal of Biological Chemistry195320412652812-s2.0-77049139172OuyangJ.ViolaR. E.Use of structural comparisons to select mutagenic targets in aspartate-β-semialdehyde dehydrogenaseBiochemistry19953419639463992-s2.0-0029041303BlancoJ.MooreR. A.FaehnleC. R.CoeD. M.ViolaR. E.The role of substrate-binding groups in the mechanism of aspartate-β-semialdehyde dehydrogenaseActa Crystallographica D2004608138813952-s2.0-1664437424410.1107/S0907444904012971SkarzńskiT.MoodyP. C. E.WonacottA. J.Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 Å resolutionJournal of Molecular Biology198719311711872-s2.0-0023644310KishM. M.ViolaR. E.Oxyanion specificity of aspartate-β-semialdehyde dehydrogenaseInorganic Chemistry19993848188202-s2.0-0001132661FaehnleC. R.BlancoJ.ViolaR. E.Structural basis for discrimination between oxyanion substrates or inhibitors in aspartate-β-semialdehyde dehydrogenaseActa Crystallographica D20046012232023242-s2.0-2164448721810.1107/S0907444904026411RaoS. T.RossmannM. G.Comparison of super secondary structures in proteinsJournal of Molecular Biology19737622412562-s2.0-0015834475ViolaR. E.LiuX.OhrenJ. F.FaehnleC. R.The structure of a redundant enzyme: a second isoform of aspartate β-semialdehyde dehydrogenase in Vibrio choleraeActa Crystallographica D20086433213302-s2.0-4044914135610.1107/S0907444907068552BiellmannJ. F.EidP.HirthC.JornvallH.Aspartate-β-semialdehyde dehydrogenase from Escherichia coli. Affinity labeling with the substrate analogue L-2-amino-4-oxo-5-chloropentanoic acid: an example of half-site reactivityEuropean Journal of Biochemistry1980104159642-s2.0-0018854054BiellmannJ. F.EidP.HirthC.Affinity labeling of the Escherichia coli aspartate-β-semialdehyde dehydrogenase with an alkylating coenzyme analogue. Half-site reactivity and competition with the substrate alkylating analogueEuropean Journal of Biochemistry1980104165692-s2.0-0018894188GaoG.LiuX.PavlovskyA.ViolaR. E.Identification of selective enzyme inhibitors by fragment library screeningJournal of Biomolecular Screening20101510421050


Archive for terrorism

What Does Islam Say about Terrorism?

Condemnation of terrorist act committed by Umar Farouk Abdulmutallab

All Muslims must condemn terrorist attacks. Terrorism is not condoned by Islam under any circumstances; and it violates the principles of justice and compassion that Islam represents.

The condemnation of terrorism is deeply rooted in true Islamic values based on the Qur’anic instructions which consider the unjust killing of a single person equivalent to the killing of all humanity (Quran, 5:32 ). It also forbids destruction and mischief on earth (Quran, 28:77,83). This violence is contrary to the authentic universal core message of peace, love, tolerance and mutual co-operation taught by Islam and all of the God’s Prophets.


Sri C.M Goenka

Principal Message

Sri C.M Goenka popularly known as 'Babuji' to many believes that moral and spiritual values need to be imbibed in children to make them responsible and enlightened citizens.

His vision is to mould every child with knowledge (JNANA) and a scientific spirit (VIJNANA). He believes that a child endowed with both Jnana and Vijnana would eventually make INDIA strong.

'Babuji' believes that we should lay adequate emphasis on our 'Sanskaras' that allows every child access to higher values of our ancient tradition and culture. He believes in providing a value based education system from the very beginning when the child's mind is clear, tender and more receptive to new learning. In his own words, "If a child is educated but unaware of one's own culture and tradition, then it is not a complete education".

"Babuji" spends much of his personal time in social service. He has started a few schools in Bangalore to provide education for the children living in slums. Quite often he visits rural and other backward areas to help the poor and the needy. He is generally found feeding and clothing the poor and needy.

J.V.V.P is the brain child of 'Babuji'. He has put ideas and energy creatively at JVVP to give a platform to his vision.

Smt. Laxmi Devi Goenka

Principal Message

Smt. Laxmi Devi Goenka shoulders the responsibility of developing JVVP into one of the best schools in the city.

She is the spirit behind the ideals of 'Babuji', our founder.

Her ambition is to implement the ideals of our founder and to prepare the children to face the challenges of the world with confidence. She believes in inculcating discipline coupled with learning among children. She is concerned about the well being of every staff member of the school.

Other Trustees:

  • Sri. Chouthmal Goenka, Chairman
  • Smt. Laxmi Devi Goenka
  • Sri. Siddharth Goenka


For those who like relaxing by the pool, there is an outdoor pool complex right next to the beach.

For the fun and laughter of your little ones there is a shallow swimming pool with a slide near the children's club and children's playground.

While your little ones have fun, you can enjoy newly renovated sunbeds and comfortable sun loungers right next to the pool.

If you are a sports person and you prefer a more active vacation, you will enjoy football, tennis, volleyball and many other sports activities.

If you want to exercise and meditate, our entertainment team will help you make the best of it through yoga practice and water aerobics.

1. All guests must shower and pass through the shower room before entering the swimming pool area.

2. Only guests of the campsite and froom the motor homes may use the swimming pool, and only with the access card.

3. Only swimmers can use the large swimming pool.

4. The swimming pool for children is only for small children and children who can't swim.

5. Parental supervision is mandatory.

6. Taking photos and videos is allowed with the permission of the staff.

7. The administration reserves the right to remove from the premises any person behaving in an improper manner and not in accordance with the Code of Conduct.

8. The administration is not responsible for items forgotten or lost.

9. Guests use the swimming pools at their own risk – the administration is not responsible for any accidents.

10. The guest is responsible for all damages.

11. Please write down all your suggestions, complaints, remarks and praise into the book at the campsite Straško reception desk.

1. Glass items and beach apperal are not allowed.

2. It is not permitted to enter the swimming pool area in footwear, not to bring in food and drinks.

3. Persons with infectious diseases may not use the swimming pools.

4. Pushing, running around, jumping and diving are not permitted.

5. Non-swimmers and small children may not use the swimming pools.

6. Pets are not allowed.

7. Persons under the influence of alcohol may not use the swimming pools.

8. Diving and pushing others into the pool, distracting other swimmers, arguing or fighting is not allowed.

9. Urinating in the swimming pools is not permitted.

10. Running on the pool deck is not allowed.

Swimming camps, shoes, goggles, a bathing suit and a towel are desirable for health, hygienic, practical, sports and other reasons!


The journey continues on the rock faces of Sink's Canyon with the sport climbers of Lander, WY and then heads north with an introduction of our editor and bearded leader. Cartter's ability to eat, jump, and get lost in a story will show you why Dirtty Toes has two t's in it.


Institute for Justice releases ‘electioneering communications’ study

Too often, campaign finance regulations are regarded as incomprehensible and inapplicable to most people’s daily lives. However, the Institute for Justice recently completed a study concerning the actual effects of “electioneering communications” restrictions on non-profit organizations and grassroots groups, and the results are troublesome to people everywhere who value First Amendment political rights.

By conducting a survey of registered non-profit organizations in Florida — which held the dubious distinction of having the broadest electioneering communications prohibition in the country (a federal district court recently struck down the law as unconstitutional) — the study aims to measure the actual impact of these laws on the day-to-day operations of small organizations.

The results are noteworthy — the reporting and disclosure requirements in these laws would, according to the non-profit survey participants, negatively affect their ability to fundraise (and, by extension, operate), communicate with members about relevant issues and more generally take valuable time away from the core missions of the groups.

The report thoroughly explains the results and is well worth the read, not to mention timely given the Supreme Court’s recent non-decision in the Citizens United case.  In fact, the study underscores the broader implications and harm these restrictive and sweeping laws inflict on core political speech.

Beyond that, however, there are several other key points that are worth emphasizing.

First, this research wisely focuses on small non-profit and grassroots groups. Too often individuals and groups who are active in politics are portrayed as exclusively big money donors and well connected PACs. This report, however, mentions several groups that would fall under these types of regulations that one would not expect – for example, a libertarian college club in Florida with an operating budget of under $100 a year, or an all volunteer group of homeowners in a community that publishes a monthly newsletter. These are not who the so-called reform community typically mentions when advocating aggressive campaign finance regulation (i.e., First Amendment limitations), but they are exactly who is affected

Secondly, there is a fundamental flaw in the logic that the more relevant an issue is to a campaign, the more government should restrict citizen’s ability to speak on it. A government that both funds political speech — as they do with publicly funded campaigns in several states and proponents hope to do with congressional elections with the Fair Elections Now Act — and also decides what issues can be discussed and by whom poses a grave threat to our democratic process. Instead of this idea of government-managed political speech, it’s imperative that citizens can associate with each other and communicate in myriad ways with the larger community about issues that most affect them, which oftentimes are issues politicians like to avoid addressing. In theory, campaigns are supposed to be a time for citizens to decide who best represents their interests in government, and not simply a regulated amount of time in which politicians offer their constituents carefully constructed sound bites.

These arguments aren’t simply theoretical. As the report notes, many states have both extremely high monetary penalties for violating electioneering communications laws, in addition to criminal penalties. Campaign finance laws are often looked upon as so obscure and complicated that most citizens wouldn’t think that they would need to consult an attorney before printing handouts at Kinko’s for a local ballot issue, for example. But in many states, they do, and failure to comply could land them in jail. There needs to be stronger language than a “chilling effect,” to describe the danger that poses to citizen engagement.

This report addresses an often overlooked but exceedingly relevant aspect of campaign finance law, noting that “states that have passed these laws have made no effort to demonstrate the existence of any compelling state interest in regulating and discouraging political electioneering communications that fall short of express advocacy.”

To date, lawmakers in fifteen states have felt no need to explain to their constituents why their ability to address relevant political issues needs government interference and criminal penalties. That state legislators no longer even feel compelled to exaggerate reasons as to why these laws need to exist should alarm any concerned about robust and free political debate.


About the Course

Enhance your employees' productivity while deepening their skills and knowledge in precision engineering with the Precision Engineering Master Craftsman (PeMC) programme.

Launched by Nanyang Polytechnic (NYP) with support from EDB, SPRING Singapore and WDA, the programme helps employees stay relevant by enhancing their technical capabilities and training them to be higher achievers.  Employees are armed with the skills and knowledge they need to support their company's drive towards higher productivity and competitiveness - essentials for success in today's global PE landscape.

What You'll Study

  • Certification

    A statement of Attainment is awarded by WDA upon successful completion of each module. The WSQ DPE MCS and the WSQ SDPE MCS are awarded when participants attain competences in the 11 modules and the 6 modules respectively.


Hi, I'm Sachin.

I've written 125+ essays sharing lessons learned from over a decade here in Silicon Valley as a product manager and startup founder.

Meet Notejoy, a better way to organize team docs

Today I’m excited to announce the launch of the productivity app my co-founder Ada and I have been building over the last 2 years. Meet Notejoy, a better way to organize team docs.

The Hierarchy of User Friction

As product designers we spend a lot of time trying to understand user friction and solve for it in the products we build. Doing so is absolutely critical to delivering delightful experiences for our users. I find though that sometimes teams are only perceiving and solving the most basic forms of user friction and aren't taking on some of the harder to perceive yet incredibly important higher level forms of friction that users are experiencing. So I wanted to share how I think about the hierarchy of user friction and provide examples and best practices for solving for each.

User friction is really anything that prevents a user from accomplishing a goal in your product. I categorize user friction into a hierarchy of three levels: interaction friction, cognitive friction, and emotional friction. Interaction friction is what I hear talked about most often amongst product designers, but the higher levels of cognitive friction and emotional friction are equally important to solve for to build a great user experience.

Top 75+ Resources for Product Managers

I'm often asked what's the best way for a new product manager to learn the fundamentals of the role or for an experienced product manager to continue to master their craft. Most folks are looking for a pointer to a book or a class they can take on product management, but I always reply with a collection of blog posts from practitioners sharing their best practices. I still believe these remain the very best resources on the topic. So I wanted to share the collection of posts I've curated over the years from incredible practitioners, writers, and thought leaders across the industry both in and outside of product roles.

I've organized this collection into several sections, starting with product management 101. I then break down the resources into the way I think about what a product manager does, which is drive the vision, strategy, design, and execution of their product. Each section covers the best practices for each of these four dimensions of product management. I then have a section on product leadership, which is important for all product managers, but especially for senior folks looking to advance in their career. And finally, I include a set of resources for managing a career in product management. In order to provide a comprehensive resource, I've included a few of my best posts at the bottom of each section.

The best way to take advantage of this collection is to dedicate 10-20 minutes each day to read through a post or two and work your way through the whole collection. Once you have you'll undoubtedly have a deep understanding of the role and what it takes to be a great product manager.

5 Keys to Unlocking Your Inner Beast Mode

I've been spending a lot of time lately thinking through how to optimize my personal productivity to drive the greatest effectiveness. In doing so I've reflected upon how there have been specific times in my life where I'm completely in the state of flow, accomplishing far more than I've ever accomplished, and absolutely loving every minute of it. And at the same time there have been days where it just feels like I'm doing sloppy work and can't seem to get my ideas to click. And I've thought about what was so special about the former days and what could I do to have far more of them. I call this unlocking my inner beast mode and I've been experimenting with a variety of practices as I pursue my latest startup to drive maximum productivity. Here are five best practices that I've discovered so far that work well for me and hopefully may be helpful for you as well.

I'm a firm believer in needing to find your personal passion/skill/opportunity fit as the ultimate path to career fulfillment. Only when you find a career that you're passionate about, that you can also be skilled at, and one that has sufficient market opportunity will you achieve ultimate career satisfaction. And it takes a careful balance of all three dimensions because simply finding fit along one dimension unfortunately won't result in success nor fulfillment.

While most folks found value in this framework when thinking about career fulfillment, some wondered what was a viable process by which they could actually get there. They knew that the role they were in wasn't it, but also didn't know how to find their personal passion/skill/opportunity fit nor how to start heading in the right direction.

To provide my perspective on this, I wanted to share my own career journey and how I specifically leveraged an explore & exploit algorithm at every turn of my career to ultimately find my dream job.


What is cybersecurity? Cybersecurity is a pretty broad term, and it refers to all of the technologies, operational procedures, and planning that makes up a system of protection intended to protect your systems and vital data from breaches or…
What is an information technology audit? An information technology audit takes a look at your existing technology infrastructure and systems, seeing what you have, what you lack, and what needs to be put in place to continue to serve your business…
A business continuity plan should be a common sense document that addresses the specific circumstances and needs of your business.
More and more,
CFO's must cultivate a deep understanding of where this vital information is at all times. How it is secured. Who might want to steal it. And most important, how they might gain access to it.


'Sweet Guardian' at Podium, Oslo, September 29th - October 15th, 2017

Traffic tacks. Cross-from-to.

Snake-squalls. It continues. Mutation-Recounts. Trivia.

Night Screen :: white lines.

This is what it tells itself:

They gather around an autonomous two-dimensional snake game, unfolding inside of an abstracted Petri dish. The snake’s meandering movement and eventual collision with its peers are recorded, examined and translated into unadulterated knowledge, systematically reforming an alphabet out of loose bits of line, mass, and void.

Randomness is their sweet guardian, caressing them gently with its abundance of extra fingers. It lives somewhere between an unknowable, divine force and a legitimized pseudo-subjectivity; the serpentine coil that brings their weather scrambles their emotions and drives their cosmos.

The exhibition comprises of a two-channel generative video, sound, wall drawings and performance.


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the Simons Foundation
and member institutions
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Physics > Atomic Physics

Title: Fast dynamical decoupling of the Molmer-Sorensen entangling gate

Abstract: Engineering entanglement between quantum systems often involves coupling through a bosonic mediator, which should be disentangled from the systems at the operation's end. The quality of such an operation is generally limited by environmental and control noise. One of the prime techniques for suppressing noise is by dynamical decoupling, where one actively applies pulses at a rate that is faster than the typical time scale of the noise. However, for boson-mediated gates, current dynamical decoupling schemes require executing the pulses only when the boson and the quantum systems are disentangled. This restriction implies an increase of the gate time by a factor of $\sqrt{N}$, with $N$ being the number of pulses applied. Here we propose and realize a method that enables dynamical decoupling in a boson mediated system where the pulses can be applied while spin-boson entanglement persists, resulting in an increase in time that is at most a factor of $\frac{\pi}{2}$, independently of the number of pulses applied. We experimentally demonstrate the robustness of our fast dynamically decoupled entangling gate to $\sigma_z$ noise with ions in a Paul trap.
Journal reference: Phys. Rev. Lett. 119, 220505 (2017)
DOI: 10.1103/PhysRevLett.119.220505
Cite as: arXiv:1706.03468 [physics.atom-ph]
  (or arXiv:1706.03468v1 [physics.atom-ph] for this version)

Submission history

From: Tom Manovitz [view email]
[v1] Mon, 12 Jun 2017 05:05:36 GMT (285kb,D)


Occupy the Future: Re-envisioning economics in an ecological world

I’d like to invite you, dear reader, into a little thought experiment, if you would. Start by taking a deep breathe. Feel the air enter through your nostrils and travel down into your lungs. Stop reading for a second and just bring your awareness to that breathe, to the process of breathing that unites you with all other beings on this earth.

Who can be said to own the air which you just breathed? It seems a ludicrous question, but imagine a world in which clean air was a commodity that was bought and sold on the open market. A world where corporations effectively owned the rights to provide clean air to paying customers.

If this seems a completely unimaginable scenario, let’s think about it a little more. Already access to clean water, no less essential to life than clean air, is being privatized and sold as a commodity in many parts of the world. As clean water becomes ever more scarce due to industrial pollution and climate change, this is a trend that will most likely accelerate. It has long been accepted that food, another of our most basic survival needs, is distributed through market exchange; and ownership of the food supply has now even been extended to the genetic code of plants.

Is it now that hard to imagine a world where air too is a commodity? What kind of world would this be? How would this affect the way we relate to one another and to the rest of the living world if even the most fundamental act of existence – breathing – was regulated by the market?

Behold the commodification of the earth, a process begun several centuries ago with the enclosure of the commons and the structuring of a rigid system of private property. This process has been so written into our dominant cultural story in the industrialized world that its implications go completely unnoticed. It is the myth of progress, of human exceptionalism, of the rationalization and institutionalization of our reality.

But this story is beginning to change. Though it is the story being told by those with power, by the mainstream media, by our various cultural and political institutions, it grows increasingly apparent how far the chasm between this official story and the daily lived experience of billions of people. And as more people begin to hear the voice of the non-human world louder and more clearly, this story becomes evermore outdated and insufficient to deal with the problems now facing our planet.

This past weekend, I and several of my colleagues at Schumacher College ran a workshop at Occupy London’s Big Green Day entitled “Occupy the Future: Re-envisioning Economics in an Ecological World.” The theme of Big Green Day was to put economic inequality and instability into the context of the larger ecological world, a perspective that has not found as much voice in the global Occupy Movement just yet.

We began to explore several deep inquiries in our short workshop: how the dominant cultural narrative constrains our understanding of the type of future that is both possible and desirable; how humans are just one species in a vastly complex, interconnected and self-organizing system called Earth; and how to begin cultivating a vision of a sustainable, equitable, meaningful future based on a co-created alternative cultural story.

We found that the process of imagining life twenty years into the future (one potential future based on the shared values of our group) was an extremely empowering experience. Once we had begun to look at ‘the economy’ as one small part of the ecological whole, inherently constrained by the boundaries of the earth’s biocapacity, the vision that evolved was one of a human-scale, decentralized society moving to the rhythms of the biosphere; it was a future of hard but meaningful work, a future of participation and abundance.

Underlying the story we began to tell was a fundamentally different relationship to the land, one characterized by a deep appreciation for life in all in manifold forms. This co-evolving relationship was one of partnership with nature, not ownership or subservience. Indeed, the exclusively anthropocentric concept of private ownership found a very limited place in this story. Once we had collectively recognized the intrinsic value of the natural world, speaking of nature in terms of commodities seemed like a particular form of violence.

If that all sounds too utopian to you, its perhaps because most of us are not exposed to the many manifestations of this vision that are already being lived and created. In Transition initiatives across the globe, communities are already making the shift to more resilient local economies; the Slow Food movement is cultivating a vision of life at a more natural pace; cooperative business ventures like Mondragon in Spain are re-envisioning structures of ownership and demonstrating that there are viable alternatives in business; organizations like the Biomimicry Institute and Permaculture Institute are evolving ways to learn from and live with the nature world; places of learning such as Barefoot College and Earth University in India are co-creating truly sustainable models of education, and movements such as Free Skools and Democratic Education are empowering people to play a more participatory role in their own learning; time banks in places such as Rushey Green are validating people’s connections outside of the monetary economy; groups such as Embercombe and The Natural Step are nurturing a new model of visionary leadership; even governments like Bhutan are embracing a more holistic understanding of human wellbeing and cultural flourishing. And of course, the Occupy movement is demonstrating the power of direct participatory decision-making.

It is certainly true that the story being told by an increasing number of people across the globe is changing far more quickly than that of our institutions. But such is the nature of change that when a certain threshold is reached (in CO2 concentrations, in cultural values, or in consciousness) a rapid shift is impossible to stop.

Given the vision of a different future that a small group of strangers created in just over an hour on a cold  London evening last weekend, it seems to me that to be a pessimist is to deny the inherent uncertainty of the universe and the limitless potential of the future. As Arundhati Roy so eloquently said, “Another world is not only possible, she is on her way. On a quiet day, I can hear her breathing.”

Ecocide: Giving name to violence against the earth

Joe Hall of Eradicating Ecocide on the case for making Ecocide the 5th international crime against peace. Speaking at Occupy London on 18 December as part of The Big Green Day,  a day of events highlighting the links between social and environmental injustice.

Eradicating Ecocide is an open-source, grassroots campaign. What are your thoughts about making ecocide an international crime? Does it address the systemic causes of environmental destruction? How might we as individuals get involved to support this cause? Share your thoughts if you fancy…

On energy systems and worldviews

According to a new report from the International Energy Agency (IEA), the world has just 5 years in which to radically transform its carbon-intensive energy infrastructure before the “lock-in” effect will make runaway climate change inevitable. A rather somber and urgent warning from one of the world’s most respected – and conservative – energy bodies.

Given the scale and design of our industrial energy infrastructure, high-carbon energy generation – coal plants, oil refineries, natural gas fracking wells, and “unconventional” tar sands mining – will continue to emit large quantities of CO² for many years after they are built. This “lock-in” effect means that the emissions of already existing or newly built carbon-burning energy sources will persist in the environment for years – centuries in fact – even once switched off. All of this makes the recent debate over whether to further exploit dirtier, more carbon-intensive forms of energy such as tar sands all the more ludicrous.

The message from scientists and energy experts could not be clearer: according to Fatih Birol, chief economist at the International Energy Agency, “if we don’t change direction now on how we use energy, we will end up beyond what scientists tell us is the minimum [for safety]. The door will be closed forever.”

An opportune time then, for the global Occupy Movement to focus on the links between a violent, unsustainable economic system and the continued exploitation and pollution of our environment. On Friday the general assembly of Occupy LSX reached preliminary consensus on an official acknowledgment of this link, adding a tenth point concerning climate change to the Occupy LSX Initial Statement. Along with the recent commitment to installing renewable energy sources such as solar panels and bicycle generators at the St. Paul’s camp, these actions go a long way towards emphasizing that it is the same underlying worldview which results in increasing concentration of wealth, corruption of the democratic process, and degradation of the one earth which all else relies upon.

People are starting to question and speak out against this entrenched economic materialism in increasing numbers. In a major victory, sustained non-violent actions against the Keystone XL pipeline in the US and Canada has resulted in the Obama administration delaying approval of the pipeline until at least next year, effectively killing the project according to Bill McKibben and 350.org. A network of activists and ordinary citizens worried about the health affects of fracking are currently organizing a large protest in New Jersey on November 21 to prevent fracking in the Delaware River Basin, the source of drinking water for 16 million people. And of course, the global Occupy Movement has galvanized action against the pervasive corporate influence that has so entrenched the fossil-fuel and finance industries perpetrating these crimes against the earth.

A solidarity action in front of the US Embassy in London protesting the Keystone XL pipeline (Nov 6)

What the mainstream media misses though, is that opposition to the Keystone XL pipeline and other similar projects is not simply about avoiding potential environmental and economic disasters like oil spills; it’s about preventing inevitable environmental and social disasters like irreversible climate change and increased privatization of the commons. Even if the pipeline does its job perfectly, ‘safely’ transporting crude oil from the Canadian tar sands to the US gulf coast, in simply consuming the resulting oil we will knowingly and consciously do more harm to the environment than any single oil spill could ever do.

It is the fundamental set of assumptions behind this pipeline – that the earth is for our exploitation, that increasing consumption will lead to higher wellbeing, that neoliberal economic policies are what is best for all – that people are beginning to reject in critical numbers.

The fact that this pipeline, like all other carbon-intensive energy projects, is painted as a savior for unemployed workers proves how untenable our current economic system really is. Yes, millions of people desperately need jobs – but not ones that will systematically destroy job opportunities and the potential to flourish for all future generations. In fact, the rapacious and extractive capitalist economy which depends on these types of massive projects necessitates systemic unemployment to keep labor costs down and maintain a surplus of workers willing to compete for any task offered to them.

Ultimately, long-term solutions designed to prevent climate change and restore the environment are also the only long-term solution to the systemic unemployment embedded in our industrial economy. And more crucially, they are solutions that restore the intrinsic dignity and meaning of work.

The transition to local, distributed, and restorative energy systems will not only drastically reduce carbon emissions and reduce environmental degradation, it will also redistribute economic and political power away from corporations and large-scale bureaucracies to communities and bioregions. Already several Transition Towns across the UK, including an ongoing project where I live in Totnes, have funded and built their own community power stations using entirely renewable energy sources. This simultaneously frees communities from dependence on fossil fuels and gives them complete autonomy over their energy supply.  The installation and maintenance of small-scale, community-owned energy systems also keeps jobs and money in local communities.

What we are talking about is not simply a matter of reconfiguring our energy infrastructure or reforming the financial system though. In the words of Joanna Macy, this is about shifting from an industrial growth society to a life sustaining society – a shift in values which places people and planet over profits and recognizes the intrinsic value of all life. This is the shift at the heart of the Occupy Movement because it represents the deepest challenge to the status quo. When our energy systems (and all other social systems) reflect a different, life-sustaining worldview, we will live in a much more just and healthier world.


Following up on the gorgeous (and epically titled) A.H.A.T.H.H.A.F.T.C.T.T.C.O.T.E., True Widow take their sound in a more propulsive direction with Circumambulation, the Texan trio's debut for Relapse Records. While True Widow are still working in their signature blend of stoner metal, drone, and shoegaze, they change the formula up with an increase in tempo. While not exactly fast, there's a certain momentum to the album that's hard to ignore, especially given the somewhat glacial pacing of their earlier work. By always ...

Circumambulation 2013, Relapse Records

UPC: 781676721825