Abstract
BackgroundSerine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.ResultsHere, 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.ConclusionsThis research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.
Highlights
Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity
The SHMT is coded by the glyA genes and act as the first enzyme in the assimilation of C1 compounds through the addition of formaldehyde to glycine, producing the principal intermediate in the pathway, serine [4]
The crystal structures of SHMT from the human, rabbit and Escherichia coli (E. coli) are reported, little is known at present about the structure-function relationship among these enzymes [5,6,7]
Summary
Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity. L-serine production mainly relies on enzymatic conversion from glycine precursor plus a C1 compound [3], and the key enzyme in L-serine enzymatic conversion is SHMT. It is necessary to obtain a SHMT with high activity of some new microorganisms and improve the catalytic efficiency through in vitro directed evolution. Structure-based sitedirected mutagenesis is usually applied to produce variants with dramatically improved specificities and the residues in or near the active site tend to be chosen as their special roles in the activity of the enzymes [21,22,23]
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