-Amino acid aminotransferase: fragmentation at a flexible loop is an efficient method to generate mutant enzymes with new substrate specificities and elevated activities

-Amino acid aminotransferase ( -AAT) (EC 2.6.1.21) catalyzes the interconversion between various -amino acids and -keto acids. A subunit of the homodimeric enzyme from a thermophile, Bacillus sp. YM-1, consists of two distinct structural domains connected by one loop. We previously constructed an active fragmentary enzyme whose backbone was cut at the interdomain loop [J. Biochem. 124 (1998) 905]. In this work, we constructed 13 fragmentary -amino acid aminotransferase genes by inserting a termination codon, an SD sequence, and an initiation codon into the specific positions of the gene corresponding to various loop regions and expressed in Escherichia coli cells. We have obtained six genes producing active fragmentary enzymes, one producing an inactive fragmentary enzyme, four producing only large peptide fragment, and another two that gave no products. The six active fragmentary enzymes purified to near-homogeneity showed various substrate specificities and thermostabilities distinct from each other and also from the wild-type enzyme: two exhibited higher catalytic activity towards -alanine, the most efficient substrate, than the wild-type enzyme. These results suggest that cleavage at a loop region is an efficient method for the alteration of enzyme properties.     

Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase.

The activity and substrate specificity of D-amino acid aminotransferase (D-AAT) (EC 2.6.1.21) can be rationally modulated by replacing the loop core (P119-R120-P121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wild-type enzyme in the overall reactions between various amino acids and pyruvate. The presteady-state kinetic analyses of half-reactions revealed that the 5-glycine mutant has the highest affinity (Kd) among all mutant enzymes and the wild-type enzyme towards various amino acids except D-aspartate. The 5-glycine mutant was much more efficient as a catalyst than the wild-type enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except D-aspartate and D-glutamate. The kmax/Kd values of the three mutants decreased with decrease in glycine chain length for each amino acid examined. Our findings may provide a new approach to rational modulation of enzymes.     

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids

Summary Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.     

Fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus: its construction and reconstitution of active fragmentary enzyme

X-ray crystallographic studies revealed that various amino acid dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an NAD(P)(+)-binding domain (Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., and Rice, D. W. (1995) Structure 3, 693-705). To elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD(+)-binding domain. The two peptide fragments were expressed in separate host cells and purified. When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared. This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of l-leucine, l-isoleucine, and l-valine with broad substrate specificity compared to that of the wild-type enzyme. The fragmentary enzyme retained more than 75% of the initial activity after heating at 50 degrees C for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site.     

In vitro characterization of the inhibitor resistance of glutamate-1-semialdehyde aminotransferase from the cyanobacterium Synechococcus PCC630l GR6

Glutamate-l-semialdehyde (GSA) aminotransferase catalyses the final step in the C5 pathway converting glutamate to the tetrapyrrole precursor δ-aminolaevulinic acid. This enzyme is sensitive to gabaculine (2,3-dihydro-3-amino benzoic acid) and to 4-amino-5-fluoropentanoic acid (AFPA), which are irreversible, mechanism-based inhibitors of pyridoxal phosphatedependent enzymes. Spontaneous mutants of Synechococcus PCC6301 resistant to these inhibitors contain altered enzyme that displays corresponding resistance to high concentrations of the inhibitor. The enzyme from strain GR6, resistant to both inhibitors, contains a three-amino-acid deletion at positions 5–7 and a Met²⁴⁸ → Ile substitution. The enzyme from strain K40 resistant to AFPA but not to gabaculine, contains a Ser¹⁶³ → Thr substitution. GSA aminotransferases containing either the deletion or the substitution that are characteristic of the GR6 mutant were produced in Escherichia coli using the expression vector pMalc2. These engineered mutant enzymes were characterized in terms of their catalytic parameters and sensitivities to gabaculine and AFPA. Furthermore, maltose binding protein/aminotransferase fusion proteins were characterized spectrophotometrically to monitor the interaction of bound cofactor with diamino- and dioxocompounds related to the substrate and both inhibitors. Results were compared with those for similarly produced recombinant wild-type, K40 and GR6 GSA aminotransferases. The engineered products with either the N-terminal deletion or the Met²⁴⁸ → Ile substitution displayed catalytic efficiencies that were intermediate between the wild-type and GR6 or K40 enzymes. However, with respect to their absorption spectra, sensitivity to inhibitors and the reactivity of bound cofactor, they were essentially wild-type. These in vitro studies demonstrate that both changes in enzyme structure are necessary to obtain the distinctive properties of the GR6 aminotransferase, including resistance to high concentrations of gabaculine and AFPA.     

Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.

Two aminotransferases from Escherichia coli were purified to homogeneity by the criterion of gel electrophoresis. The first (enzyme A) is active on L-aspartic acid, L-tyrosine, L-phenylalanine, and L-tryptophan; the second (enzyme B) is active on the aromatic amiono acids. Enzyme A is identical in substrate specificity with transaminase A and is mainly an aspartate aminotransferase; enzyme B has never been described before and is an aromatic amino acid aminotransferase. The two enzymes are different in the Vmax and Km values with their common substrates and pyridoxal phosphate, in heat stability (enzyme A being heat-stable and enzyme B being heat-labile at 55 degrees) and in pH optima with the amino acid substrates. They are similar in their amino acid composition, each enzyme appears to consist of two subunits, and enzyme B may be converted to enzyme A by controlled proteolysis with subtilsin. The conversion was detected by the generation of new aspartate aminotransferase activity from enzyme B and was further verified by identification by acrylamide gel electrophoresis of the newly formed enzyme A. The two enzymes appear to be products of two genes different in a small, probably terminal, nucleotide sequence.     

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.     

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.     

Reconstitution of Fragmentary Form of Thermostable Alanine Racemase

The fragmentary form of alanine racemase from Bacillus stearothermophilus is composed of two sets of two different polypeptides corresponding to the two domains of the subunit of wild-type enzyme. It was denatured with 6 M guanidium hydrochloride to be separated into pieces, and renatured by dilution with about 50% recovery of the activity. The two kinds of polypeptides (i.e., large and a small fragments) were isolated by gel filtration in the presence of 4 M guanidium hydrochloride. The CD spectra obtained by summation of the spectra of the refolded fragments were closely similar to that of the native fragmentary enzyme. The lysine residue to which PLP is bound in the wild-type enzyme occurs in the large peptide of the fragmentary enzyme containing the amino terminus of the wild-type enzyme. The visible spectrum of the large peptide refolded, however, indicated that PLP was not bound to it. The large peptide alone had no significant activity, but it was activated by incubation with the small peptide. Accordingly, co-existene of both peptide fragments is necessary for folding of a complex of the two kinds of peptide to form an active structure.     

<Emphasis Type="Italic">Thermobifida fusca</Emphasis> exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.     

Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.

Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli. One mutation, designated tyrB, lies at about 80 min on the E. coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase. The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids. In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis. The ilvE gene product alone can reverse a phenylalanine requirement. Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate. These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility. In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.     

l-amino acid oxidases of Proteus rettgeri.

Proteus rettgeri has been found to contain two separable 1-amino acid oxidases. Both enzymes are particulate in nature, neither being ribosomal bound. One of these enzymes appears to have broad specificity, being active toward monoaminomonocarboxylic, imino, aromatic, sulfur-containing, and beta-hydroxyamino acids. The other enzyme has more limited specificity, catalyzing the oxidative deamination of the basic amino acids and citrulline. The affinity of this oxidase for the various substrates at pH 7.6 in decreasing order is arginine, histidine, ornithine, citrulline, and lysine. This enzyme has a particularly high affinity for arginine (Km equal to 0.27 mM), and anomalous kinetics are observed with increasing substrate concentrations. When concentrations of arginine greater than 1.0mM were added to the reaction containing histidine, imidazole pyruvate formation was completely inhibited.     

Computational Selection,Identification and Structural Analysis of ω-Aminotransferases with Various Substrate Specificities from the Genome Sequence of Mesorhizobium loti MAFF303099

ω-Aminotransferase (ω-AT) is an important class of enzymes for the synthesis of chiral amines or β-amino acids. Family profile analysis was applied to screen putative ω-ATs from Mesorhizobium loti MAFF303099, a nitrogen fixation bacterium that has a larger number of ATs than other microorganisms. By family profile analysis, we selected 10 putative ω-ATs according to E-value. The functions of the putative ω-ATs were investigated by examining activities towards amines and/or β-amino acids. 10 putative proteins were found to have ω-AT activity with narrow or broad substrate specificity. Structure analysis using crystal structure of mll7127 and homology models of mll1632 and mll3663 indicated that the structures of active sites of the enzymes were very similar and highly conserved, but their substrate specificities appreared to be determined by residues positioned at the entrance region of the active site binding pockets.     

Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme

Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora

Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.     

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp²³⁰ residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.     

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.     

Effect of Feeding Bengal Gram (Cicer arietinum) at Different Protein Levels on Plasma Lipids of Rats

L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.     

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.