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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

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A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic

Wei Jiang¹, Lin Chen², Nan Hu³

¹State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, P. R. China. tianya416@126.com.

BMC Biotechnology

|November 15, 2014

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.

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