NORMAL GENOMIC

• Singh A, Kushwaha HR, Sharma P
• Molecular modelling and comparative structural account of aspartylbeta-semialdehyde dehydrogenase of Mycobacterium tuberculosis (H37Rv).
• J Mol Model. 2008; 14: 249-63
• Display abstract

• Aspartyl beta-semialdehyde dehydrogenase (ASADH) is an important enzyme,occupying the first branch position of the biosynthetic pathway of theaspartate family of amino acids in bacteria, fungi and higher plants. Itcatalyses reversible dephosphorylation of L: -beta-aspartyl phosphate(betaAP) to L: -aspartate-beta-semialdehyde (ASA), a key intermediate inthe biosynthesis of diaminopimelic acid (DAP)-an essential component ofcross linkages in bacterial cell walls. Since the aspartate pathway isunique to plants and bacteria, and ASADH is the key enzyme in thispathway, it becomes an attractive target for antimicrobial agentdevelopment. Therefore, with the objective of deducing comparativestructural models, we have described a molecular model emphasizing theuniqueness of ASADH from Mycobacterium tuberculosis (H37Rv) that shouldgenerate insights into the structural distinctiveness of this protein ascompared to structurally resolved ASADH from other bacterial species. Wefind that mtASADH exhibits structural features common to bacterial ASADH,while other structural motifs are not present. Structural analysis ofvarious domains in mtASADH reveals structural conservation among allbacterial ASADH proteins. The results suggest that the probable mechanismof action of the mtASADH enzyme might be same as that of other bacterialASADH. Analysis of the structure of mtASADH will shed light on itsmechanism of action and may help in designing suitable antagonists againstthis enzyme that could control the growth of Mycobacterium tuberculosis.

• Viola RE, Liu X, Ohren JF, Faehnle CR
• The structure of a redundant enzyme: a second isoform of aspartatebeta-semialdehyde dehydrogenase in Vibrio cholerae.
• Acta Crystallogr D Biol Crystallogr. 2008; 64: 321-30
• Display abstract

• Aspartate-beta-semialdehyde dehydrogenase (ASADH) is an essential enzymethat is found in bacteria, fungi and plants but not in humans. ASADHproduces the first branch-point metabolite in the biosynthetic pathwaysthat lead to the production of lysine, threonine, methionine andisoleucine as well as the cell-wall precursor diaminopimelate. As aconsequence, ASADH appears to be an excellent target for the developmentof novel antibiotics, especially for Gram-negative bacteria that requirediaminopimelate for cell-wall biosynthesis. In contrast to theGram-negative ASADHs, which readily formed well diffracting crystals, thesecond isoform of aspartate-beta-semialdehyde dehydrogenase from Vibriocholerae (vcASADH2) was less well behaved in initial crystallizationtrials. In order to obtain good-quality single crystals of vcASADH2, abuffer-optimization protocol was used in which the initial purificationbuffer was exchanged into a new condition derived from a pre-crystallinehit. The unliganded structure of vcASADH2 has been determined to 2.2 Aresolution to provide additional insight into the structural andfunctional evolution of the ASADH enzyme family. The overall fold anddomain organization of this new structure is similar to the Gram-negative,Gram-positive and archeal ASADH structures determined previously, despitehaving less than 50% sequence identity to any of these family members. Thesubstrate-complex structure reveals that the binding ofL-aspartate-beta-semialdehyde (ASA) to vcASADH2 is accommodated bystructural changes in the amino-acid binding site and in the helicalsubdomain that is involved in the dimer interface. Structural alignmentsshow that this second isoform from Gram-negative V. cholerae most closelyresembles the ASADH from a Gram-positive organism and is likely to bindthe coenzyme in a different conformation to that observed in the other V.cholerae isoform.

• Nichols CE, Dhaliwal B, Lockyer M, Hawkins AR, Stammers DK
• High-resolution structures reveal details of domain closure and"half-of-sites-reactivity" in Escherichia coli aspartate beta-semialdehydedehydrogenase.
• J Mol Biol. 2004; 341: 797-806
• Display abstract

• Two high-resolution structures have been determined for Eschericia coliaspartate beta-semialdehyde dehydrogenase (ecASADH), an enzyme of theaspartate biosynthetic pathway, which is a potential target for novelantimicrobial drugs. Both ASADH structures were of the open form and wererefined to 1.95 A and 1.6 A resolution, allowing a more detailedcomparison with the closed form of the enzyme than previously possible. Amore complex scheme for domain closure is apparent with the subunit beingsplit into two further sub-domains with relative motions about three hingeaxes. Analysis of hinge data and torsion-angle difference plots iscombined to allow the proposal of a detailed structural mechanism forecASADH domain closure. Additionally, asymmetric distortions of individualsubunits are identified, which form the basis for the previously reported"half-of-the-sites reactivity" (HOSR). A putative explanation of thisarrangement is also presented, suggesting the HOSR system may provide ameans for ecASADH to offset the energy required to remobilise flexibleloops at the end of the reaction cycle, and hence avoid falling into anenergy minimum.

• Blanco J, Moore RA, Kabaleeswaran V, Viola RE
• A structural basis for the mechanism of aspartate-beta-semialdehydedehydrogenase from Vibrio cholerae.
• Protein Sci. 2003; 12: 27-33
• Display abstract

• L-Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes thereductive dephosphorylation of beta-aspartyl phosphate toL-aspartate-beta-semialdehyde in the aspartate biosynthetic pathway ofplants and micro-organisms. The aspartate pathway produces fullyone-quarter of the naturally occurring amino acids, but is not found inhumans or other eukaryotic organisms, making ASADH an attractive targetfor the development of new antibacterial, fungicidal, or herbicidalcompounds. We have determined the structure of ASADH from Vibrio choleraein two states; the apoenzyme and a complex with NADP, and a covalentlybound active site inhibitor, S-methyl-L-cysteine sulfoxide. Upon bindingthe inhibitor undergoes an enzyme-catalyzed reductive demethylationleading to a covalently bound cysteine that is observed in the complexstructure. The enzyme is a functional homodimer, with extensiveintersubunit contacts and a symmetrical 4-amino acid bridge linking theactive site residues in adjacent subunits that could serve as acommunication channel. The active site is essentially preformed, withminimal differences in active site conformation in the apoenzyme relativeto the ternary inhibitor complex. The conformational changes that do occurresult primarily from NADP binding, and are localized to the repositioningof two surface loops located on the rim at opposite sides of the NADPcleft.

• Blanco J, Moore RA, Viola RE
• Capture of an intermediate in the catalytic cycle ofL-aspartate-beta-semialdehyde dehydrogenase.
• Proc Natl Acad Sci U S A. 2003; 100: 12613-7
• Display abstract

• The structural analysis of an enzymatic reaction intermediate affords aunique opportunity to study a catalytic mechanism in extraordinary detail.Here we present the structure of a tetrahedral intermediate in thecatalytic cycle of aspartate-beta-semialdehyde dehydrogenase (ASADH) fromHaemophilus influenzae at 2.0-A resolution. ASADH is not found in humans,yet its catalytic activity is required for the biosynthesis of essentialamino acids in plants and microorganisms. Diaminopimelic acid, also formedby this enzymatic pathway, is an integral component of bacterial cellwalls, thus making ASADH an attractive target for the development of newantibiotics. This enzyme is able to capture the substratesaspartate-beta-semialdehyde and phosphate as an active complex that doesnot complete the catalytic cycle in the absence of NADP. A distinctivebinding pocket in which the hemithioacetal oxygen of the bound substrateis stabilized by interaction with a backbone amide group dictates the Rstereochemistry of the tetrahedral intermediate. This pocket, reminiscentof the oxyanion hole found in serine proteases, is completed throughhydrogen bonding to the bound phosphate substrate.

• Hadfield A et al.
• Active site analysis of the potential antimicrobial target aspartatesemialdehyde dehydrogenase.
• Biochemistry. 2001; 40: 14475-83
• Display abstract

• Aspartate-beta-semialdehyde dehydrogenase (ASADH) lies at the first branchpoint in the biosynthetic pathway through which bacteria, fungi, and thehigher plants synthesize amino acids, including lysine and methionine andthe cell wall component diaminopimelate from aspartate. Blocks in thisbiosynthetic pathway, which is absent in mammals, are lethal, andinhibitors of ASADH may therefore serve as useful antibacterial,fungicidal, or herbicidal agents. We have determined the structure ofASADH from Escherichia coli by crystallography in the presence of itscoenzyme and a substrate analogue that acts as a covalent inhibitor. Thisstructure is comparable to that of the covalent intermediate that formsduring the reaction catalyzed by ASADH. The key catalytic residues areconfirmed as cysteine 135, which is covalently linked to the intermediateduring the reaction, and histidine 274, which acts as an acid/basecatalyst. The substrate and coenzyme binding residues are also identified,and these active site residues are conserved throughout all of the ASADHsequences. Comparison of the previously determined apo-enzyme structure[Hadfield et al. J. Mol. Biol. (1999) 289, 991-1002] and the complexpresented here reveals a conformational change that occurs on binding ofNADP that creates a binding site for the amino acid substrate. Theseresults provide a structural explanation for the preferred order ofsubstrate binding that is observed kinetically.

• Scapin G, Reddy SG, Blanchard JS
• Three-dimensional structure of meso-diaminopimelic acid dehydrogenase fromCorynebacterium glutamicum.
• Biochemistry. 1996; 35: 13540-51
• Display abstract

• Diaminopimelate dehydrogenase catalyzes the NADPH-dependent reduction ofammonia and L-2-amino-6-ketopimelate to form meso-diaminopimelate, thedirect precursor of L-lysine in the bacterial lysine biosynthetic pathway.Since mammals lack this metabolic pathway inhibitors of enzymes in thispathway may be useful as antibiotics or herbicides. Diaminopimelatedehydrogenase catalyzes the only oxidative deamination of an amino acid ofD configuration and must additionally distinguish between two chiral aminoacid centers on the same symmetric substrate. The Corynebacteriumglutamicum enzyme has been cloned, expressed in Escherichia coli, andpurified to homogeneity using standard biochemical procedures [Reddy, S.G., Scapin, G., & Blanchard, J. S. (1996) Proteins: Structure, Funct.Genet. 25, 514-516]. The three-dimensional structure of the binary complexof diaminopimelate dehydrogenase with NADP+ has been solved using multipleisomorphous replacement procedures and noncrystallographic symmetryaveraging. The resulting model has been refined against 2.2 A diffractiondata to a conventional crystallographic R-factor of 17.0%. Diaminopimelatedehydrogenase is a homodimer of structurally not identical subunits. Eachsubunit is composed of three domains. The N-terminal domain contains amodified dinucleotide binding domain, or Rossman fold (six centralbeta-strands in a 213456 topology surrounded by five alpha-helices). Thesecond domain contains two alpha-helices and three beta-strands. Thisdomain is referred to as the dimerization domain, since it is involved informing the monomer--monomer interface of the dimer. The third orC-terminal domain is composed of six beta-strands and five alpha-helices.The relative position of the N- and C-terminal domain in the two monomersis different, defining an open and a closed conformation that mayrepresent the enzyme's binding and active state, respectively. In bothmonomers the nucleotide is bound in an extended conformation across theC-terminal portion of the beta-sheet of the Rossman fold, with its C4facing the C-terminal domain. In the closed conformer two molecules ofacetate have been refined in this region, and we postulate that theydefine the DAP binding site. The structure of diaminopimelatedehydrogenase shows interesting similarities to the structure of glutamatedehydrogenase [Baker, P. J., Britton, K. L., Rice, D. W., Rob, A., &Stillmann, T.J. (1992a) J. Mol. Biol. 228, 662-671] and leucinedehydrogenase [Baker, P.J., Turnbull, A.P., Sedelnikova, S.E., Stillman,T. J., & Rice, D. W. (1995) Structure 3, 693-705] and also resembles thestructure of dihydrodipicolinate reductase [Scapin, G., Blanchard, J. S.,& Sacchettini, J. C. (1995) Biochemistry 34, 3502-3512], the enzymeimmediately preceding it in the diaminopimelic acid/lysine biosyntheticpathway.

• Ouyang J, Viola RE
• Use of structural comparisons to select mutagenic targets inaspartate-beta-semialdehyde dehydrogenase.
• Biochemistry. 1995; 34: 6394-9
• Display abstract

• L-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) from Escherichia colihas been probed by site-directed mutagenesis to identify residues thatplay an important function in the catalytic activity of the enzyme.Sequence homology searching among ASA DHs that have been isolated fromother species and comparisons with the structures of functionally similarD-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) that have been solvedfrom several species have been utilized to select appropriate targets formutagenesis. A highly conserved active site glutamine has been identifiedin the E. coli ASA DH that enhances the reactivity of the enzyme.Alteration of this residue leads to an enzyme with reduced catalyticefficiency, yet with an unchanged binding affinity for substrates andcoenzyme. Replacement of an arginine residue that is conserved throughoutthe ASA DH and GAPDH enzyme families leads to a significant decrease incatalytic turnover and is the only mutation examined that also results ina decreased affinity for the substrates of the reaction. This residue isassigned a role in the binding of the substrateaspartate-beta-semialdehyde. Sequence alignment of ASA DH with other NADP-and NAD-dependent enzymes has resulted in the identification of a putativepyridine nucleotide binding region. Substitution of two amino acids inthis region with neutral or positively charged side chains has resulted ina change in enzyme specificity. For wild-type ASA DH, NADP is stronglyfavored as the coenzyme, while in this mutated enzyme the selectivity hasbeen lowered by a factor of 60, and this enzyme has comparable affinitiesfor either pyridine nucleotide.

• Karsten WE, Viola RE
• Identification of an essential cysteine in the reaction catalyzed byaspartate-beta-semialdehyde dehydrogenase from Escherichia coli.
• Biochim Biophys Acta. 1992; 1121: 234-8
• Display abstract

• The enzyme L-aspartate-beta-semialdehyde dehydrogenase from Escherichiacoli has been studied by oligonucleotide-directed mutagenesis. The focusof this investigation was to examine the role of a cysteine residue thathad been previously identified by chemical modification with an activesite directed reagent (Biellmann et al. (1980) Eur. J. Biochem. 104,59-64). Substitution of this cysteine at position 135 with an alanineresults in complete loss of enzyme activity. However, changing thiscysteine to a serine yields a mutant enzyme with a maximum velocity thatis 0.3% that of the native enzyme. This C135S mutant has retainedessentially the same affinity for substrates as the native enzyme, and thesame overall conformation as reflected in identical behavior on gelelectrophoresis and in identical fluorescence spectra. The pH profile ofthe native enzyme shows a loss in catalytic activity upon protonation of agroup with a pKa value of 7.7. The same activity loss is observed at thispH with the serine-135 mutant, despite the differences in the pKa valuesfor a cysteine sulfhydryl and a serine hydroxyl group that have beenmeasured in model compounds. This observed pKa value may reflect theprotonation of an auxiliary catalyst that enhances the reactivity of theactive site cysteine nucleophile in the native aspartate-beta-semialdehydedehydrogenase.

• Biellmann JF, Eid P, Hirth C, Jornvall H
• Aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.Purification and general properties.
• Eur J Biochem. 1980; 104: 53-8
• Display abstract

• Aspartate-beta-semialdehyde dehydrogenase, from an Escherichia coli mutantderepressed for the biosynthesis of L-lysine, has been purified tohomogeneity. Its isoelectric point is pH 4.3. This enzyme has a molecularweight of 77000 and is composed of two identical or highly similarsubunits of molecular weight 38000 +/- 2000. Their N-terminal amino-acidsequence is Met-Lys-Asx-Val-Gly-. Three cysteine residues per subunit weredetected: two are reactive in the native enzyme and one is partiallyprotected by the substrate. Formation of an acyl-enzyme intermediate wasalso detected. Correlation of the 1H nuclear magnetic resonance spectrumof [4-2H]NADPH produced from [4-2H]NADP+ indicated that aspartatebeta-semialdehyde dehydrogenase transfers the pro-S hydrogen from NADPH(class B dehydrogenase). A short comparison with the corresponding yeastenzyme is given.