Structural-functional analysis of drug target aspartate semialdehyde dehydrogenase.

Aspartate ß-semialdehyde dehydrogenase (ASADH) is a key enzyme in the biosynthesis of essential amino acids in microorganisms and some plants. Inhibition of ASADHs can be a potential drug target for developing novel antimicrobial and herbicidal compounds. This review covers up-to-date information about sequence diversity, ligand/inhibitor-bound 3D structures, potential inhibitors, and key pharmacophoric features of ASADH useful in designing novel and target-specific inhibitors of ASADH. Most reported ASADH inhibitors have two highly electronegative functional groups that interact with two key arginyl residues present in the active site of ASADHs. The structural information, active site binding modes, and key interactions between the enzyme and inhibitors serve as the basis for designing new and potent inhibitors against the ASADH family.

Molecular evolution, binding site interpretation and functional divergence of aspartate semialdehyde dehydrogenase.

Aspartate Semialdehyde Dehydrogenase (ASDH) is an important enzyme essential for the viability of pathogenic microorganisms. ASDH is mainly involved in amino acid and cell wall biosynthesis of microorganisms, hence it is considered to be a promising target for drug design. This enzyme depicts similar mechanistic function in all microorganisms; although, the kinetic efficiency of an enzyme differs according to their active site residual composition. Therefore, understanding the residual variation and kinetic efficiency of the enzyme would pave new insights in structure-based drug discovery and a novel drug molecule against ASDH. Here, ASDH from Wolbachia endosymbiont of Brugia malayi is used as a prime enzyme to execute evolutionary studies. The phylogenetic analysis was opted to classify 400 sequences of ASDH enzymes based on their structure and electrostatic surfaces. Analysis resulted in 37 monophyletic clades of diverse pathogenic and non-pathogenic organisms. The representative structures of 37 ASDHs from different clades were further deciphered to structural homologues. These enzymes exhibited presence of more positively charged surfaces than negatively charged surfaces in the active site pocket which restrains evolutionary significance. Docking studies of NADP+ with 37 enzymes reveals that site-specific residual variation in the active site pocket modulates the binding affinity (ranges of -13 to -9 kcal/mol). Type-I and Type-II divergence studies show, no significant functional divergence among ASDH, but residual changes were found among the enzyme that modulates the biochemical characteristics and catalytic efficiency. The present study not only explores residual alteration and catalytic variability, it also aids in the design of species-specific inhibitors.Communicated by Ramaswamy H. Sarma.

IMB-XMA0038, a new inhibitor targeting aspartate-semialdehyde dehydrogenase of Mycobacterium tuberculosis.

The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programmes. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC50 value of 0.59 µg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25-0.5 µg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10-5 or 10-1 µg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.

Identification of anti-filarial leads against aspartate semialdehyde dehydrogenase of Wolbachia endosymbiont of Brugia malayi: combined molecular docking and molecular dynamics approaches.

Lymphatic filariasis is a debilitating vector borne parasitic disease that infects human lymphatic system by nematode Brugia malayi. Currently available anti-filarial drugs are effective only on the larval stages of parasite. So far, no effective drugs are available for humans to treat filarial infections. In this regard, aspartate semialdehyde dehydrogenase (ASDase) in lysine biosynthetic pathway from Wolbachia endosymbiont Brugia malayi represents an attractive therapeutic target for the development of novel anti-filarial agents. In this present study, molecular modeling combined with molecular dynamics simulations and structure-based virtual screening were performed to identify potent lead molecules against ASDase. Based on Glide score, toxicity profile, binding affinity and mode of interactions with the ASDase, five potent lead molecules were selected. The molecular docking and dynamics results revealed that the amino acid residues Arg103, Asn133, Cys134, Gln161, Ser164, Lys218, Arg239, His246, and Asn321 plays a crucial role in effective binding of Top leads into the active site of ASDase. The stability of the ASDase-lead complexes was confirmed by running the 30 ns molecular dynamics simulations. The pharmacokinetic properties of the identified lead molecules are in the acceptable range. Furthermore, density functional theory and binding free energy calculations were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-filarial agents to combat the pathogenecity of Brugia malayi.

Structural insights into inhibitor binding to a fungal ortholog of aspartate semialdehyde dehydrogenase.

The aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors. The first inhibitor-bound and cofactor-bound structures of ASADH from the pathogenic fungi Blastomyces dermatitidis have now been determined, along with a structural and functional comparison to other ASADH family members. The structure of this new ASADH is similar to the other fungal orthologs, but with some critical differences in the orientation of some active site functional groups and in the subunit interface region. The presence of this bound inhibitor reveals the first details about inhibitor binding interactions, and the flexible orientation of its aromatic ring provides helpful insights into the design of potentially more potent and selective antifungal compounds.

Structure of aspartate ß-semialdehyde dehydrogenase from Francisella tularensis.

Aspartate ß-semialdehyde dehydrogenase (ASADH) is an enzyme involved in the diaminopimelate pathway of lysine biosynthesis. It is essential for the viability of many pathogenic bacteria and therefore has been the subject of considerable research for the generation of novel antibiotic compounds. This manuscript describes the first structure of ASADH from Francisella tularensis, the causative agent of tularemia and a potential bioterrorism agent. The structure was determined at 2.45 Šresolution and has a similar biological assembly to other bacterial homologs. ASADH is known to be dimeric in bacteria and have extensive interchain contacts, which are thought to create a half-sites reactivity enzyme. ASADH from higher organisms shows a tetrameric oligomerization, which also has implications for both reactivity and regulation. This work analyzes the apo form of F. tularensis ASADH, as well as the binding of the enzyme to its cofactor NADP+.

Aspartate-ß-semialdeyhyde dehydrogenase as a potential therapeutic target of Mycobacterium tuberculosis H37Rv: Evidence from in silico elementary mode analysis of biological network model.

The emergence of multi-drug resistant strains and co-occurrence of tuberculosis with HIV creates a major burden to the human health globally. Failure of primary antibacterial therapy necessitates the identification of new mycobacterial drugs. In this study, a comprehensive analysis involving bottom-up systems biology approach was applied wherein we have identified potential therapeutic targets of Mycobacterium tuberculosis infections. Our study prioritized M. tuberculosis therapeutic targets (aspartate-ß-semialdeyhde dehydrogenase [ASD], dihydrodipicolinate reductase and diaminopimelate decarboxylase) based on flux and elementary mode analysis using direct mathematical modeling of the relevant metabolic pathways. Molecular docking and simulation studies of the priority target (ie, ASD) revealed the therapeutic potential of the selected natural products (Huperzine A, Rosmarinic acid, and Curcumin) based ASD inhibitors. The study highlights the crucial role of systems biology in conjunction with molecular interaction (docking) for probing novel leads against an increasingly resistant pathogen, M. tuberculousis.

Structure of a fungal form of aspartate-semialdehyde dehydrogenase from Aspergillus fumigatus.

Aspartate-semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of ß-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. The absence of this entire pathway in humans and other mammals will allow the selective targeting of pathogenic microorganisms for antimicrobial development. Here, the X-ray structure of a new form of ASADH from the pathogenic fungal species Aspergillus fumigatus has been determined. The overall structure of this enzyme is similar to those of its bacterial orthologs, but there are some critical differences both in biological assembly and in secondary-structural features that can potentially be exploited for the development of species-selective drugs with selective toxicity against infectious fungal organisms.

Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site.

The gene encoding a quinoprotein aldose sugar dehydrogenase (ASD) from Thermus thermophilus HJ6 (Tt_ASD) was cloned and sequenced; it comprised 1059 nucleotides encoding a protein containing 352 amino acids that had a predicted molecular mass of 38.9 kDa. The deduced amino acid sequence showed 42.9% and 33.9% identities to the ASD proteins from Pyrobaculum aerophilum and Escherichia coli, respectively. The biochemical properties of Tt_ASD were characterized. The optimum pH for the oxidation of glucose was 7.0-7.5 and the optimum temperature was 70 °C. The half-life of heat inactivation for the apoenzyme was about 25 min at 85 °C. The enzyme was highly thermostable, and the activity of the pyrroloquinoline quinone-bound holoenzyme was not lost after incubation at 85 °C for 100 min. Tt_ASD could oxidize various sugars, including hexoses, pentoses, disaccharides, and polysaccharides, in addition to alcohols. Structural analysis suggested that Tyr156 would be the substrate-binding residue. Two mutants, Y156A and Y156K, had impaired activities and affinities for all substrates and completely lost their activities for alcohols. This structural and mutational analysis of Tt_ASD demonstrates the crucial role of Tyr156 in determining substrate specificity.

Structural Insights into the Tetrameric State of Aspartate-ß-semialdehyde Dehydrogenases from Fungal Species.

Aspartate-ß-semialdehyde dehydrogenase (ASADH) catalyzes the second reaction in the aspartate pathway, a pathway required for the biosynthesis of one fifth of the essential amino acids in plants and microorganisms. Microarray analysis of a fungal pathogen T. rubrum responsible for most human dermatophytoses identified the upregulation of ASADH (trASADH) expression when the fungus is exposed to human skin, underscoring its potential as a drug target. Here we report the crystal structure of trASADH, revealing a tetrameric ASADH with a GAPDH-like fold. The tetramerization of trASADH was confirmed by sedimentation and SAXS experiments. Native PAGE demonstrated that this ASADH tetramerization is apparently universal in fungal species, unlike the functional dimer that is observed in all bacterial ASADHs. The helical subdomain in dimeric bacteria ASADH is replaced by the cover loop in archaeal/fungal ASADHs, presenting the determinant for this altered oligomerization. Mutations that disrupt the tetramerization of trASADH also abolish the catalytic activity, suggesting that the tetrameric state is required to produce the active fungal enzyme form. Our findings provide a basis to categorize ASADHs into dimeric and tetrameric enzymes, adopting a different orientation for NADP binding and offer a structural framework for designing drugs that can specifically target the fungal pathogens.

Pharmacoinformatics analysis to identify inhibitors of Mtb-ASADH.

Aspartate-semialdehyde dehydrogenase (ASADH; EC 1.2.1.11) is a key enzyme in the biosynthesis of essential amino acids in prokaryotes and fungi, inhibition of ASADH leads to the development of novel antitubercular agents. In the present work, a combined structure and ligand-based pharmacophore modeling, molecular docking, and molecular dynamics (MD) approaches were employed to identify potent inhibitors of mycobacterium tuberculosis (Mtb)-ASADH. The structure-based pharmacophore hypothesis consists of three hydrogen bond acceptor (HBA), two negatively ionizable, and one positively ionizable center, while ligand-based pharmacophore consists of additional one HBA and one hydrogen bond donor features. The validated pharmacophore models were used to screen the chemical databases (ZINC and NCI). The screened hits were subjected to ADME and toxicity filters, and subsequently to the molecular docking analysis. Best-docked 25 compounds carry the characteristics of highly electronegative functional groups (-COOH and -NO2) on both sides and exhibited the H-bonding interactions with highly conserved residues Arg99, Arg249, and His256. For further validation of docking results, MD simulation studies were carried out on two representative compounds NSC51108 and ZINC04203124. Both the compounds remain bound to the key active residues of Mtb-ASADH during the MD simulations. These identified hits can be further used for lead optimization and in the design more potent inhibitors against Mtb-ASADH.

Structure of a fungal form of aspartate semialdehyde dehydrogenase from Cryptococcus neoformans.

Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate-biosynthetic pathway and represents a valid target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of ß-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step in the biosynthesis of the essential amino acids methionine, isoleucine and threonine in fungi, and also the amino acid lysine in bacteria. The structure of a new fungal form of ASADH from Cryptococcus neoformans has been determined to 2.6 Å resolution. The overall structure of CnASADH is similar to those of its bacterial orthologs, but with some critical differences both in biological assembly and in secondary-structural features that can potentially be exploited for the development of species-selective drugs.

Shape-based virtual screening, docking, and molecular dynamics simulations to identify Mtb-ASADH inhibitors.

Aspartate ß-semialdehyde dehydrogenase (ASADH) is a key enzyme for the biosynthesis of essential amino acids and several important metabolites in microbes. Inhibition of ASADH enzyme is a promising drug target strategy against Mycobacterium tuberculosis (Mtb). In this work, in silico approach was used to identify potent inhibitors of Mtb-ASADH. Aspartyl ß-difluorophosphonate (ß-AFP), a known lead compound, was used to understand the molecular recognition interactions (using molecular docking and molecular dynamics analysis). This analysis helped in validating the computational protocol and established the participation of Arg99, Glu224, Cys130, Arg249, and His256 amino acids as the key amino acids in stabilizing ligand-enzyme interactions for effective binding, an essential feature is H-bonding interactions with the two arginyl residues at the two ends of the ligand. Best binding conformation of ß-AFP was selected as a template for shape-based virtual screening (ZINC and NCI databases) to identify compounds that competitively inhibit the Mtb-ASADH. The top rank hits were further subjected to ADME and toxicity filters. Final filter was based on molecular docking analysis. Each screened molecule carries the characteristics of the highly electronegative groups on both sides separated by an average distance of 6 Å. Finally, the best predicted 20 compounds exhibited minimum three H-bonding interactions with Arg99 and Arg249. These identified hits can be further used for designing the more potent inhibitors against ASADH family. MD simulations were also performed on two selected compounds (NSC4862 and ZINC02534243) for further validation. During the MD simulations, both compounds showed same H-bonding interactions and remained bound to key active residues of Mtb-ASADH.

A cautionary tale of structure-guided inhibitor development against an essential enzyme in the aspartate-biosynthetic pathway.

The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate ß-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme-inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.

A modified Lumry-Eyring analysis for the determination of the predominant mechanism underlying the diminution of protein aggregation by glycerol.

Aggregation of aspartate-ß-semialdehyde dehydrogenase (ASD) was analyzed by applying modified Lumry-Eyring with nucleated polymerization (LENP) model. Intrinsic nucleation time scales were determined. In absence of glycerol, ASD undergoes concentration and time-dependent polymerization into low-molecular weight soluble aggregates and thereafter condensation into insoluble aggregates. In the presence of increasing solvent glycerol concentration, the aggregation becomes more and more nucleation dominated, with slower polymerization to low-molecular weights soluble aggregates, without any condensation into insoluble aggregates. Effective nucleus size as well as the number of monomers in each irreversible growth event were sensitive to the changes in solvent glycerol concentration. Glycerol-directed diminution of aggregation appears to be largely due to the inhibition of rearrangement (decreased nucleation rearrangement rate coefficient, K r,x ) because of compaction induced due to preferential hydration, thus, preventing the soluble aggregates from locking into irreversible soluble nuclei. Appreciably decreased K r,x (as compared to nucleation dissociation constant, K d,x ), appears to be responsible for increased nucleus size at higher solvent glycerol concentration. This study explains how modified LENP model can be applied to determine the predominant mechanism responsible for the diminution of aggregation by polyhydric alcohols (glycerol).

Structures of ternary complexes of aspartate-semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis H37Rv.

Aspartate-semialdehyde dehydrogenase (Asd; ASADH; EC 1.2.1.11) is the enzyme that lies at the first branch point in the biosynthetic pathway of important amino acids including lysine and methionine and the cell-wall component diaminopimelate (DAP). The enzymatic reaction of ASADH is the reductive dephosphorylation of aspartyl-ß-phosphate (ABP) to aspartate ß-semialdehyde (ASA). Since the aspartate pathway is absolutely essential for the survival of many microbes and is absent in humans, the enzymes involved in this pathway can be considered to be potential antibacterial drug targets. In this work, the structure of ASADH from Mycobacterium tuberculosis H37Rv (Mtb-ASADH) has been determined in complex with glycerol and sulfate at 2.18 Å resolution and in complex with S-methyl-L-cysteine sulfoxide (SMCS) and sulfate at 1.95 Å resolution. The overall structure of Mtb-ASADH is similar to those of its orthologues. However, in the Mtb-ASADH-glycerol complex structure the glycerol molecule is noncovalently bound to the active-site residue Cys130, while in the Mtb-ASADH-SMCS complex structure the SMCS (Cys) is covalently linked to Cys130. The Mtb-ASADH-SMCS complex structurally mimics one of the intermediate steps in the proposed mechanism of ASADH enzyme catalysis. Comparison of the two complex structures revealed that the amino acids Glu224 and Arg249 undergo conformational changes upon binding of glycerol. Moreover, the structures reported here may help in the development of species-specific antibacterial drug molecules against human pathogens.

Expansion of the aspartate beta-semialdehyde dehydrogenase family: the first structure of a fungal ortholog.

The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a critical transformation that produces the first branch-point intermediate in an essential microbial amino-acid biosynthetic pathway. The first structure of an ASADH isolated from a fungal species (Candida albicans) has been determined as a complex with its pyridine nucleotide cofactor. This enzyme is a functional dimer, with a similar overall fold and domain organization to the structurally characterized bacterial ASADHs. However, there are differences in the secondary-structural elements and in cofactor binding that are likely to cause the lower catalytic efficiency of this fungal enzyme. Alterations in the dimer interface, through deletion of a helical subdomain and replacement of amino acids that participate in a hydrogen-bonding network, interrupt the intersubunit-communication channels required to support an alternating-site catalytic mechanism. The detailed functional information derived from this new structure will allow an assessment of ASADH as a possible target for antifungal drug development.

The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology.

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.

Purification, crystallization and preliminary X-ray diffraction analysis of aspartate semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis.

Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B.

The structure of a redundant enzyme: a second isoform of aspartate beta-semialdehyde dehydrogenase in Vibrio cholerae.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) is an essential enzyme that is found in bacteria, fungi and plants but not in humans. ASADH produces the first branch-point metabolite in the biosynthetic pathways that lead to the production of lysine, threonine, methionine and isoleucine as well as the cell-wall precursor diaminopimelate. As a consequence, ASADH appears to be an excellent target for the development of novel antibiotics, especially for Gram-negative bacteria that require diaminopimelate for cell-wall biosynthesis. In contrast to the Gram-negative ASADHs, which readily formed well diffracting crystals, the second isoform of aspartate-beta-semialdehyde dehydrogenase from Vibrio cholerae (vcASADH2) was less well behaved in initial crystallization trials. In order to obtain good-quality single crystals of vcASADH2, a buffer-optimization protocol was used in which the initial purification buffer was exchanged into a new condition derived from a pre-crystalline hit. The unliganded structure of vcASADH2 has been determined to 2.2 A resolution to provide additional insight into the structural and functional evolution of the ASADH enzyme family. The overall fold and domain organization of this new structure is similar to the Gram-negative, Gram-positive and archeal ASADH structures determined previously, despite having less than 50% sequence identity to any of these family members. The substrate-complex structure reveals that the binding of L-aspartate-beta-semialdehyde (ASA) to vcASADH2 is accommodated by structural changes in the amino-acid binding site and in the helical subdomain that is involved in the dimer interface. Structural alignments show that this second isoform from Gram-negative V. cholerae most closely resembles the ASADH from a Gram-positive organism and is likely to bind the coenzyme in a different conformation to that observed in the other V. cholerae isoform.