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A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

ABSTRACT: BACKGROUND: Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity. RESULTS: Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40 °C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum's SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position. CONCLUSIONS: This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

SUBMITTER: Jiang W

PROVIDER: S-EPMC4260256 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

Jiang Wei W Chen Lin L Hu Nan N Yuan Shaohui S Li Bin B Liu Ziduo Z

BMC biotechnology 20141114

<h4>Background</h4>Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.<h4>Results</h4>Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40 °C and pH 7.5, and was more stable in weakly alkali conditions (pH 6. ...[more]

PMID: 25394480

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Project description:BackgroundTrehalases have potential applications in several fields, including food additives, insecticide development, and transgenic plant. In the present study, we focused on a trehalase from the marine bacterium Zunongwangia sp., which hydrolyzes trehalose to glucose.ResultsA novel gene, treZ (1590 bp) encoding an α, α-trehalase of 529 amino acids was cloned from Zunongwangia sp., and TreZ was found to have an optimal activity at 50 °C and pH 6. The activity of TreZ was increased by the presence of NaCl, showing the highest activity (136 %) at 1 M NaCl. A variant C4 with improved catalytic activity was obtained by error-prone PCR and followed by a 96-well plate high-throughput screening. The variant C4 with two altered sites (Y227H, and R442G) displayed a 3.3 fold increase in catalytic efficiency (k cat/K m, 1143.40 mmol(-1) s(-1)) compared with the wild type enzyme (265.91 mmol(-1) s(-1)). In order to explore the contribution of the mutations found in variant C4 to the increased catalytic activity, two mutants Y227H and R442G were constructed by site-directed mutagenesis. The results showed that the catalytic efficiencies of Y227H and R442G were 416.78 mmol(-1) s(-1) and 740.97 mmol(-1) s(-1), respectively, indicating that both mutations contributed to the increased catalytic efficiency of variant C4. The structure modeling and substrate docking revealed that the substitution Y227H enlarged the shape of the binding pocket, to improve the binding of the substrate and the release of the products; while the substitution R442G reduced the size of the side chain and decreased the steric hindrance, which contributed to channel the substrate into the active cavity easier and promote the release of the product.ConclusionIn this study, a novel trehalase was cloned, purified, characterized, and engineered. A variant C4 with dramatically improved catalytic activity was obtained by directed evolution, and the mutation sites Y227H and R442G were found to play a significant role in the catalytic efficiency. The overall results provide useful information about the structure and function of trehalase.

Dataset Information

A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

Publications

A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

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