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

Jiang W, Chen L, Hu N, Yuan S, Li B, Liu Z - BMC Biotechnol. (2014)

Bottom Line: Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.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.This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

View Article: PubMed Central - PubMed

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

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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Related in: MedlinePlus

Phylogenetic tree of the AnSHMT. The phylogenetic tree was established using the program MEGA 5.05. The SHMT protein sequences were obtained from GenBank and PDB (http://www.rcsb.org/pdb/home/home.do), except for AnSHMT.
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Fig1: Phylogenetic tree of the AnSHMT. The phylogenetic tree was established using the program MEGA 5.05. The SHMT protein sequences were obtained from GenBank and PDB (http://www.rcsb.org/pdb/home/home.do), except for AnSHMT.

Mentions: Using degenerate primers, a fragment was obtained by DOP-PCR with the genomic DNA of A. nicotianae as the template. The open reading frame (ORF) of the glyA (1323 bp, GenBank: KF359496) fragment encoded a polypeptide of 440 amino acids, with a deduced molecular mass of 47.3 kDa. The phylogenetic analyses of the SHMT sequences produced a tree to further verify the evolutionary relationship among AnSHMT and other known SHMTs, indicating that the AnSHMT shared 54.3% amino acid identity with the known SHMT from E. coli (Figure 1).Figure 1


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

Jiang W, Chen L, Hu N, Yuan S, Li B, Liu Z - BMC Biotechnol. (2014)

Phylogenetic tree of the AnSHMT. The phylogenetic tree was established using the program MEGA 5.05. The SHMT protein sequences were obtained from GenBank and PDB (http://www.rcsb.org/pdb/home/home.do), except for AnSHMT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260256&req=5

Fig1: Phylogenetic tree of the AnSHMT. The phylogenetic tree was established using the program MEGA 5.05. The SHMT protein sequences were obtained from GenBank and PDB (http://www.rcsb.org/pdb/home/home.do), except for AnSHMT.
Mentions: Using degenerate primers, a fragment was obtained by DOP-PCR with the genomic DNA of A. nicotianae as the template. The open reading frame (ORF) of the glyA (1323 bp, GenBank: KF359496) fragment encoded a polypeptide of 440 amino acids, with a deduced molecular mass of 47.3 kDa. The phylogenetic analyses of the SHMT sequences produced a tree to further verify the evolutionary relationship among AnSHMT and other known SHMTs, indicating that the AnSHMT shared 54.3% amino acid identity with the known SHMT from E. coli (Figure 1).Figure 1

Bottom Line: Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.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.This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus