Kinetic and Crystallographic Analysis of Active Site Mutants of <i>Escherichia coli </i>γ-Aminobutyrate Aminotransferase

Liu, Wenshe Ray; Peterson, Pamela E.; Langston, James; Jin, Xing; Zhou, Xiuteng; Fisher, A.J.; Toney,

doi:10.1021/bi048657a

Cited by 44 publications

(41 citation statements)

References 25 publications

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“…The HAB aminotransferase described here showed 66% amino acid sequence identity with the annotated 4-aminobutryate aminotransferase from Pseudomonas syringae ( Table 1). The protein identified here also showed 46% amino acid sequence identity to the E. coli 4-aminobutyrate aminotransferase for which an X-ray structure is known (17).…”

Section: Discussionmentioning

confidence: 63%

Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

2008

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L-(؊)-Azetidine-2-carboxylate (AC) is a toxic, natural product analog of L-proline. This study revealed the genes and biochemical strategy employed by Pseudomonas sp. strain A2C to detoxify and assimilate AC as its sole nitrogen source. The gene region from Pseudomonas sp. strain A2C required for detoxification was cloned into Escherichia coli and sequenced. The 7.0-kb region contained eight identifiable genes. Four encoded putative transporters or permeases for ␥-amino acids or drugs. Another gene encoded a homolog of 2-haloacid dehalogenase (HAD). The encoded protein, denoted L-azetidine-2-carboxylate hydrolase (AC hydrolase), was highly overexpressed by subcloning. The AC hydrolase was shown to catalyze azetidine ring opening with the production of 2-hydroxy-4-aminobutyrate. AC hydrolase was further demonstrated to be a new hydrolytic member of the HAD superfamily by showing loss of activity upon changing aspartate-12, the conserved active site nucleophile in this family, to an alanine residue. The presence of a gene encoding a potential export chaperone protein, CsaA, adjacent to the AC hydrolase gene suggested that AC hydrolase might be found inside the periplasm in the native Pseudomonas strain. Periplasmic and cytoplasmic cell fractions from Pseudomonas sp. strain A2C were prepared. A higher specific activity for AC hydrolysis was found in the periplasmic fraction. Protein mass spectrometry further identified AC hydrolase and known periplasmic marker proteins in the periplasmic fraction. A model was proposed in which AC is hydrolyzed in the periplasm and the product of that reaction is transported into and further metabolized in the cytoplasm.Plants produce many toxins that offer protection against grazing by insects and mammals (11). Some of those toxins act by mimicking essential amino acids. One such natural product is S-(Ϫ)-azetidine-2-carboxylate (AC), which can comprise up to 7% of the leaf mass of Lily of the Valley plants (6, 7) and has more recently been found to be present in sugar beets (26). AC mimics the amino acid L-proline. Upon ingestion by a susceptible animal, AC becomes incorporated into proteins in place of L-proline. The different bond angles in AC compared to L-proline causes change in the protein backbone, which can result in disruption of function (8). AC is also produced by Streptomyces spp. and is considered to be an antibiotic, but it is too toxic to humans to be useful clinically.AC is toxic to microorganisms if it is transported into the cell and incorporated into proteins (8). However, Saccharomyces cerevisiae is known to detoxify AC via acetylation of the carboxylic acid group (32). Additionally, bacteria are known that not only tolerate AC but use it as the sole source of nitrogen (5,35,36). Bacteria growing on AC are reported to open the azetidine ring and susbsequently capture the nitrogen atom via a transamination reaction. The data are consistent with a mechanism involving an enzyme-catalyzed hydrolytic opening of the azetidine ring. However, until the present study, th...

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Section: Discussionmentioning

confidence: 63%

Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

2008

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“…This model fits well with the abundant evidence, demonstrating that, in vivo, Orn-AT operates in the direction in which ornithine and ketoglutarate are the substrates (4). Moreover, the hypothesis is supported by the recently published structure (20) of a mutant form of GABA-AT (I50Q) that is partially in the E M form and in which the homologue of Glu-235 (E211) is seen to be rotated away from the homologous conserved arginine. Furthermore, the structure of the homologue of Orn-AT-E235S (GABA-AT-E211S), in which the switch must be permanently open, shows the enzyme to be entirely in the E M form (20), reinforcing the proposal that the status of the cofactor and the Glu-235 switch are linked.…”

Section: Properties Of Mutant Forms Of Orn-at-mentioning

confidence: 87%

Determinants of Substrate Specificity in ω-Aminotransferases

Marková

Peneff

Hewlins

et al. 2005

Journal of Biological Chemistry

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Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the ␣-carboxylate of ␣-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13 C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the ␦-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.Ornithine aminotransferase (Orn-AT) 3 and 4-aminobutyrate aminotransferase (GABA-AT) are members of a large family of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes that catalyze a wide range of reactions on amino acids (1). Each enzyme operates by a mechanism, common to all aminotransferases ( Fig. 1), in which the cofactor shuttles between pyridoxaldimine and pyridoxamine forms by means of two coupled half-reactions (2, 3). The half-reaction converting ketoglutarate to glutamate is the same for both enzymes as well as for the majority of other aminotransferases. In this half-reaction, the chemical changes occur at the ␣-carbon. The substrate specificity of the enzymes thus arises from the other half-reaction that transfers an amino group distant from the ␣-carbon. For this reason, the enzymes are known as "-aminotransferases" (4). In the case of GABA-AT, the amino group transferred is the only amino group in the substrate molecule, whereas Orn-AT specifically selects the -amino group of ornithine despite the presence of a second amino group on C␣ with the same configuration as that in glutamate (3).The three-dimensional structures of both enzymes have been solved in the unliganded form as well as in complex with various substrate analogues (5-7). The enzymes have the same b...

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“…The identity and similarity of the amino acid sequences of PuuE and GabT were 54.2% and 67.4%, respectively. The sequence alignment between PuuE and GabT (data not shown) suggested that Lys-267 of PuuE (corresponding to Lys-268 in GabT, which is an important residue for GabT activity [10]) is an important residue for the activity. Site-directed mutagenesis of PuuE(K267A) was performed to confirm that Lys-267 is an important residue of PuuE.…”

Section: Fig 2 (A)mentioning

confidence: 99%

A Putrescine-Inducible Pathway Comprising PuuE-YneI in Which γ-Aminobutyrate Is Degraded into Succinate in Escherichia coli K-12

et al. 2010

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␥-Aminobutyrate (GABA) is metabolized to succinic semialdehyde by GABA aminotransferase (GABA-AT), and the succinic semialdehyde is subsequently oxidized to succinate by succinic semialdehyde dehydrogenase (SSADH). In Escherichia coli, there are duplicate GABA-ATs (GabT and PuuE) and duplicate SSADHs (GabD and YneI). While GabT and GabD have been well studied previously, the characterization and expression analysis of PuuE and YneI are yet to be investigated. By analyzing the amino acid profiles in cells of ⌬puuE and/or ⌬gabT mutants, this study demonstrated that PuuE plays an important role in GABA metabolism in E. coli cells. The similarity of the amino acid sequences of PuuE and GabT is 67.4%, and it was biochemically demonstrated that the catalytic center of GabT is conserved as an amino acid residue important for the enzymatic activity in PuuE as Lys-247. However, the regulation of expression of PuuE is significantly different from that of GabT. PuuE is induced by the addition of putrescine to the medium and is repressed by succinate and low aeration conditions; in contrast, GabT is almost constitutive. Similarly, YneI is induced by putrescine, while GabD is not. For E. coli, PuuE is important for utilization of putrescine as a sole nitrogen source and both PuuE and YneI are important for utilization of putrescine as a sole carbon source. The results demonstrate that the PuuE-YneI pathway was a putrescine-inducible GABA degradation pathway for utilizing putrescine as a nutrient source.

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Kinetic and Crystallographic Analysis of Active Site Mutants of Escherichia coli γ-Aminobutyrate Aminotransferase

Cited by 44 publications

References 25 publications

Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

Determinants of Substrate Specificity in ω-Aminotransferases

A Putrescine-Inducible Pathway Comprising PuuE-YneI in Which γ-Aminobutyrate Is Degraded into Succinate in Escherichia coli K-12

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