Inhibition of 7,8-diaminopelargonic acid aminotransferase from Mycobacterium tuberculosis by chiral and achiral anologs of its substrate: Biological implications

7,8-Diaminopelargonic acid aminotransferase (DAPA AT), a potential drug target in Mycobacterium tuberculosis, transforms 8-amino-7-oxononanoic acid (KAPA) into DAPA. We have designed an analytical method to measure the enantiomeric excess of KAPA, based on the derivatization of its amine function, by ortho-phtalaldehyde and N-acetyl-l-cysteine, followed by high pressure liquid chromatography separation. Using this methodology and enantiopure samples of KAPA it appeared that racemization of KAPA occurs rapidly (half-lives from 1 to 8 h) not only in 4 M HCl but more importantly in the usual pH range, from 7 to 9. Furthermore, we showed that racemic KAPA, and not enantiopure KAPA, was used in all previous studies. The only valid enantioselective synthesis of KAPA is that reported by Lucet et al. (1996) Tetrahedron: Asymmetry 7, 985–988. KAPA is produced as a pure (S)-enantiomer by KAPA synthase and by microbial production and DAPA AT only uses (S)-KAPA as substrate. However, (R)-KAPA is an inhibitor of this enzyme. It binds to the pyridoxal 5′-phosphate form (K_i1 = 5.9 ± 0.2 μM) and to the pyridoxamine 5′-phosphate form (K_i2 = 1.7 ± 0.2 μM) of M. tuberculosis DAPA AT. Molecular modeling showed that (R)-KAPA forms specific hydrogen bonds with T309 and the phosphate group of the cofactor of DAPA AT. Desmethyl-KAPA (8-amino-7-oxooctanoic acid), an achiral analog of KAPA, is also a potent inhibitor of M. tuberculosis DAPA AT. This molecule binds to the enzyme in a similar way than (R)-KAPA with the following constants: K_i1 = 4.2 ± 0.2 μM, and K_i2 = 0.9 ± 0.2 μM. These findings pave the way to the design of new antimycobacterial drugs.     

Mechanism of substrate recognition and PLP-induced conformational changes in LL-diaminopimelate aminotransferase from Arabidopsis thaliana

LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) and N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the alpha-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C(epsilon) amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.     

Structure of biosynthetic N-acetylornithine aminotransferase from Salmonella typhimurium: studies on substrate specificity and inhibitor binding

Acetylornithine aminotransferase (AcOAT) is one of the key enzymes involved in arginine metabolism and catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate. It belongs to the Type I subgroup II family of pyridoxal 5'-phosphate (PLP) dependent enzymes. E. coli biosynthetic AcOAT (eAcOAT) also catalyzes the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate, one of the steps in lysine biosynthesis. In view of the critical role of AcOAT in lysine and arginine biosynthesis, structural studies were initiated on the enzyme from S. typhimurium (sAcOAT). The K(m) and k(cat)/K(m) values determined with the purified sAcOAT suggested that the enzyme had much higher affinity for AcOrn than for ornithine (Orn) and was more efficient than eAcOAT. sAcOAT was inhibited by gabaculine (Gcn) with an inhibition constant (K(i)) of 7 microM and a second-order rate constant (k(2)) of 0.16 mM(-1) s(-1). sAcOAT, crystallized in the unliganded form and in the presence of Gcn or L-glutamate, diffracted to a maximum resolution of 1.90 A and contained a dimer in the asymmetric unit. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop L(alphak-) (betam) in only one subunit (subunit B; the loop from subunit B comes close to the phosphate group of PLP in subunit A). Structural and spectral studies of sAcOAT with Gcn suggested that the enzyme might have a low affinity for PLP-Gcn complex. Comparison of sAcOAT with T. thermophilus AcOAT and human ornithine aminotransferase suggested that the higher specificity of sAcOAT towards AcOrn may not be due to specific changes in the active site residues but could result from minor conformational changes in some of them. This is the first structural report of AcOAT from a mesophilic organism and could serve as a basis for drug design as the enzyme is important for bacterial cell wall biosynthesis.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k_cat′ for desulfination of l-cysteine sulfinate increased to 0.5 s^− 1 (from 0.05 s^− 1 in wild-type enzyme), whereas k_cat′ for transamination of the same substrate was reduced from 510 s^− 1 to 0.05 s^− 1. Similarly, k_cat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s^− 1 to 0.07 s^− 1, whereas k_cat′ for transamination was reduced from 530 s^− 1 to 0.13 s^− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.     

Crystal structure of LL-diaminopimelate aminotransferase from Arabidopsis thaliana: a recently discovered enzyme in the biosynthesis of L-lysine by plants and Chlamydia

The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5'-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the design of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 A resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modelled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.     

The three-dimensional structure of N-succinyldiaminopimelate aminotransferase from Mycobacterium tuberculosis

Inhibitors of the enzymes of the lysine biosynthetic pathway are considered promising lead compounds for the design of new antibacterial drugs, because the pathway appears to be indispensable for bacteria and because it is absent in humans. As part of our efforts to structurally characterize all enzymes of this pathway in Mycobacterium tuberculosis (Mtb), we have determined the three-dimensional structure of N-succinyldiaminopimelate aminotransferase (DapC, DAP-AT, Rv0858c) to a resolution of 2.0 A. This structure is the first DAP-AT structure reported to date. The orthorhombic crystals of Mtb-DAP-AT contain one functional dimer exhibiting C(2) symmetry in the asymmetric unit. The homodimer displays the typical S-shape of class I pyridoxal-5'-phosphate (PLP)-binding proteins. The two active sites of the dimer both feature an internal aldimine with the co-factor PLP covalently bound to the Lys232, although neither substrate nor co-factor had been added during protein production, purification and crystallization. Nine water molecules are conserved in the active site and form an intricate hydrogen-bonding network with the co-factor and the surrounding amino acid residues. Together with some residual difference electron density in the active site, this architecture permitted the building of external aldimine models of the enzyme with the substrates glutamate, the amine donor, and N-succinyl-2-amino-6-keto-pimelate, the amine acceptor. Based on these models, the amino acids relevant for substrate binding and specificity can be postulated. Furthermore, in the external aldimine model of N-succinyl-2-amino-6-keto-pimelate, the succinyl group overlaps with a glycerol binding site that has also been identified in both active sites of the Mtb-DAP-AT dimer. A comparison of the structure of Mtb-DAP-AT with other class I PLP-binding proteins, revealed that some inhibitors utilize the same binding site. Thus, the proposed models also provide an explanation for the mode of inhibition of Mtb-DAP-AT and they may be of help in the design of compounds, which are capable of inhibiting the enzyme. Last, but not least, a chloride binding helix exhibiting a peculiar amino acid sequence with a number of exposed hydrophobic side-chains was identified, which may be hypothesized as a putative docking site.     

Crystal structure of Saccharomyces cerevisiae Aro8, a putative α‐aminoadipate aminotransferase

α‐Aminoadipate aminotransferase (AAA‐AT) catalyzes the amination of 2‐oxoadipate to α‐aminoadipate in the fourth step of the α‐aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA‐AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2‐oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA‐AT LysN from Thermus thermophilus and human kynurenine aminotransferase II. Inspection of the active site of Aro8 reveals asymmetric cofactor binding with lysine‐pyridoxal‐5‐phosphate bound within the active site of one subunit in the Aro8 homodimer and pyridoxamine phosphate and a HEPES molecule bound to the other subunit. The HEPES buffer molecule binds within the substrate‐binding site of Aro8, yielding insights into the mechanism by which it recognizes multiple substrates and how this recognition differs from other AAA‐AT/kynurenine aminotransferases.     

Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode

The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group.     

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.     

Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases

5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK_a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.     

The crystal structure of the cytosolic exopolyphosphatase from Saccharomyces cerevisiae reveals the basis for substrate specificity

Inorganic long-chain polyphosphate is a ubiquitous linear polymer in biology, consisting of many phosphate moieties linked by phosphoanhydride bonds. It is synthesized by polyphosphate kinase, and metabolised by a number of enzymes, including exo- and endopolyphosphatases. The Saccharomyces cerevisiae gene PPX1 encodes for a 45 kDa, metal-dependent, cytosolic exopolyphosphatase that processively cleaves the terminal phosphate group from the polyphosphate chain, until inorganic pyrophosphate is all that remains. PPX1 belongs to the DHH family of phosphoesterases, which includes: family-2 inorganic pyrophosphatases, found in Gram-positive bacteria; prune, a cyclic AMPase; and RecJ, a single-stranded DNA exonuclease. We describe the high-resolution X-ray structures of yeast PPX1, solved using the multiple isomorphous replacement with anomalous scattering (MIRAS) technique, and its complexes with phosphate (1.6 A), sulphate (1.8 A) and ATP (1.9 A). Yeast PPX1 folds into two domains, and the structures reveal a strong similarity to the family-2 inorganic pyrophosphatases, particularly in the active-site region. A large, extended channel formed at the interface of the N and C-terminal domains is lined with positively charged amino acids and represents a conduit for polyphosphate and the site of phosphate hydrolysis. Structural comparisons with the inorganic pyrophosphatases and analysis of the ligand-bound complexes lead us to propose a hydrolysis mechanism. Finally, we discuss a structural basis for substrate selectivity and processivity.     

Identification of potential drugs targeting L,L-diaminopimelate aminotransferase of Chlamydia trachomatis: An integrative pharmacoinformatics approach

Chlamydia trachomatis (C.t) is a gram-negative obligate intracellular bacteria, which is a major causative of infectious blindness and sexually transmitted diseases. A surge in multidrug resistance among chlamydial species has posed a challenge to adopt alternative drug targeting strategies. Recently, in C.t, L,L-diaminopimelate aminotransferase (CtDAP-AT) is proven to be a potential drug target due its essential role in cell survival and host nonspecificity. Hence, in this study, a multilevel precision-based virtual screening of CtDAP-AT was performed to identify potential inhibitors, wherein, an integrative stringent scoring and filtration were performed by coupling, glide docking score, binding free energy, ADMET (absorption, distribution, metabolism, and excretion, toxicity) prediction, density functional theory (quantum mechanics), and molecular dynamics simulation (molecular mechanics). On cumulative analysis, NSC_5485 (1,3-bis((7-chloro-4-quinolinyl)amino)-2-propanol) was found to be the most potential lead, as it showed higher order significance in terms of binding affinity, bonded interactions, favorable ADMET, chemical reactivity, and greater stabilization during complex formation. This is the first report on prioritization of small molecules from National Cancer Institute (NCI) and Maybridge data sets (341 519 compounds) towards targeting CtDAP-AT. Thus, the proposed compound shall aid in effective combating of a broad spectrum of C.t infections as it surpassed all the levels of prioritization.     

Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis

Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.     

Direct evidence for a glutamate switch necessary for substrate recognition: crystal structures of lysine epsilon-aminotransferase (Rv3290c) from Mycobacterium tuberculosis H37Rv

Lysine epsilon-aminotransferase (LAT) is a PLP-dependent enzyme that is highly up-regulated in nutrient-starved tuberculosis models. It catalyzes an overall reaction involving the transfer of the epsilon-amino group of L-lysine to alpha-ketoglutarate to yield L-glutamate and alpha-aminoadipate-delta-semialdehyde. We have cloned and characterized the enzyme from Mycobacterium tuberculosisH37Rv. We report here the crystal structures of the enzyme, the first from any source, in the unliganded form, external aldimine with L-lysine, with bound PMP and with its C5 substrate alpha-ketoglutarate. In addition to interaction details in the active site, the structures reveal a Glu243 "switch" through which the enzyme changes substrate specificities. The unique substrate L-lysine is recognized specifically when Glu243 maintains a salt-bridge with Arg422. On the other hand, the binding of the common C5 substrates L-glutamate and alpha-ketoglutarate is enabled when Glu243 switches away and unshields Arg422. The structures reported here, sequence conservation and earlier mutational studies suggest that the "glutamate switch" is an elegant solution devised by a subgroup of fold type I aminotransferases for recognition of structurally diverse substrates in the same binding site and provides for reaction specificity.     

Molecular function prediction for a family exhibiting evolutionary tendencies toward substrate specificity swapping: Recurrence of tyrosine aminotransferase activity in the Iα subfamily

The subfamily Iα aminotransferases are typically categorized as having narrow specificity toward carboxylic amino acids (AATases), or broad specificity that includes aromatic amino acid substrates (TATases). Because of their general role in central metabolism and, more specifically, their association with liver‐related diseases in humans, this subfamily is biologically interesting. The substrate specificities for only a few members of this subfamily have been reported, and the reliable prediction of substrate specificity from protein sequence has remained elusive. In this study, a diverse set of aminotransferases was chosen for characterization based on a scoring system that measures the sequence divergence of the active site. The enzymes that were experimentally characterized include both narrow‐specificity AATases and broad‐specificity TATases, as well as AATases with broader‐specificity and TATases with narrower‐specificity than the previously known family members. Molecular function and phylogenetic analyses underscored the complexity of this family's evolution as the TATase function does not follow a single evolutionary thread, but rather appears independently multiple times during the evolution of the subfamily. The additional functional characterizations described in this article, alongside a detailed sequence and phylogenetic analysis, provide some novel clues to understanding the evolutionary mechanisms at work in this family. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Moraxella catarrhalis Lgt2, a galactosyltransferase with broad acceptor substrate specificity

The genetic basis of lipo-oligosaccharide (LOS) biosynthesis for the bacterium Moraxella catarrhalis has been elucidated and functions suggested for each of the glycosyltransferases. In this study we have expressed and characterised one of these enzymes, the putative galactosyltransferase Lgt2_B/C. The lgt2_B/C gene was amplified from M. catarrhalis, expressed in Escherichia coli, and Lgt2_B/C was purified. Analysis of its glycosyltransferase catalytic activity ascertained the pH and temperature optima. The donor specificity and acceptor specificity were examined and they showed that Lgt2_B/C is a galactosyltransferase with relatively broad acceptor specificity with optimal activity in the presence of exogenous Mg²⁺.     

Cysteine and keto acids modulate mosquito kynurenine aminotransferase catalyzed kynurenic acid production

Kynurenine aminotransferase (KAT) catalyzes the formation of kynurenic acid (KYNA), the natural antagonist of ionotropic glutamate receptors. This study tests potential substrates and assesses the effects of amino acids and keto acids on the activity of mosquito KAT. Various keto acids, when simultaneously present in the same reaction mixture, display a combined effect on KAT catalyzed KYNA production. Moreover, methionine and glutamine show inhibitory effects on KAT activity, while cysteine functions as either an antagonist or an inhibitor depending on the concentration. Therefore, the overall level of keto acids and cysteine might modulate the KYNA synthesis. Results from this study will be useful in the study of KAT regulation in other animals.     

Structural and biochemical basis for the substrate specificity of Pad-1, an indole-3-pyruvic acid aminotransferase in auxin homeostasis

Phytohormone indole-3-acetic acid (IAA) plays a vital role in regulating plant growth and development. Tryptophan-dependent IAA biosynthesis participates in IAA homeostasis by producing IAA via two sequential reactions, which involve a conversion of tryptophan to indole-3-pyruvic acid (IPyA) by tryptophan aminotransferase (TAA1) followed by the irreversible formation of IAA in the second reaction. Pad-1 from Solanaceae plants regulates IAA levels by catalyzing a reverse reaction of the first step of IAA biosynthesis. Pad-1 is a pyridoxal phosphate (PLP)-dependent aminotransferase, with IPyA as the amino acceptor and l-glutamine as the amino donor. Currently, the structural and functional basis for the substrate specificity of Pad-1 remains poorly understood. In this study, we carried out structural and kinetic analyses of Pad-1 from Solanum melongena. Pad-1 is a homodimeric enzyme, with coenzyme PLP present between a central large α/β domain and a protruding small domain. The active site of Pad-1 includes a vacancy near the phosphate group (P-side) and the 3′-O (O-side) of PLP. These features are distinct from those of TAA1, which is homologous in an overall structure with Pad-1 but includes only the P-side region in the active site. Kinetic analysis suggests that P-side residues constitute a binding pocket for l-glutamine, and O-side residues of Phe124 and Ile350 are involved in the binding of IPyA. These studies illuminate distinct differences in the active site between Pad-1 and TAA1, and provide structural and functional insights into the substrate specificity of Pad-1.