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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

Multiple sequence comparison by ClustalW. (a) a part of the sequence alignment result (81-160 sites); (b) a part of the sequence alignment result (161-240 sites); (c) a part of the sequence alignment result (241-320 sites). Open boxes indicate the four highly conserved regions and the conserved active site is underlined. The site-directed mutagenesis site is underlined and is marked by five-pointed star. The amino acid sequences were obtained from NCBI database (http://www.ncbi.nlm.nih.gov/).
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Fig2: Multiple sequence comparison by ClustalW. (a) a part of the sequence alignment result (81-160 sites); (b) a part of the sequence alignment result (161-240 sites); (c) a part of the sequence alignment result (241-320 sites). Open boxes indicate the four highly conserved regions and the conserved active site is underlined. The site-directed mutagenesis site is underlined and is marked by five-pointed star. The amino acid sequences were obtained from NCBI database (http://www.ncbi.nlm.nih.gov/).

Mentions: Multiple sequence comparisons were performed by Clustal W Method, indicating that the conserved amino acid sequences and motifs are critical for the active site of SHMT (Figure 2). The highly conserved active site in all known SHMT enzymes T/ST/STTHKT/SL was also found in AnSHMT (235–242) in the form of TSTTHKTL (Figure 2c), the putative active site [5,14,24]. The site of the site-directed mutagenesis underlined and marked by a five-pointed star was the close proximity to the highly conserved active site and the putative active site (TSTTHKTL) (Figure 2c). The deduced active site structure (TSTTHKTL) and the position of the mutant (249 isoleucine) were briefly presented the three-dimensional structure of AnSHMT (Figure 3).Figure 2


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)

Multiple sequence comparison by ClustalW. (a) a part of the sequence alignment result (81-160 sites); (b) a part of the sequence alignment result (161-240 sites); (c) a part of the sequence alignment result (241-320 sites). Open boxes indicate the four highly conserved regions and the conserved active site is underlined. The site-directed mutagenesis site is underlined and is marked by five-pointed star. The amino acid sequences were obtained from NCBI database (http://www.ncbi.nlm.nih.gov/).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Multiple sequence comparison by ClustalW. (a) a part of the sequence alignment result (81-160 sites); (b) a part of the sequence alignment result (161-240 sites); (c) a part of the sequence alignment result (241-320 sites). Open boxes indicate the four highly conserved regions and the conserved active site is underlined. The site-directed mutagenesis site is underlined and is marked by five-pointed star. The amino acid sequences were obtained from NCBI database (http://www.ncbi.nlm.nih.gov/).
Mentions: Multiple sequence comparisons were performed by Clustal W Method, indicating that the conserved amino acid sequences and motifs are critical for the active site of SHMT (Figure 2). The highly conserved active site in all known SHMT enzymes T/ST/STTHKT/SL was also found in AnSHMT (235–242) in the form of TSTTHKTL (Figure 2c), the putative active site [5,14,24]. The site of the site-directed mutagenesis underlined and marked by a five-pointed star was the close proximity to the highly conserved active site and the putative active site (TSTTHKTL) (Figure 2c). The deduced active site structure (TSTTHKTL) and the position of the mutant (249 isoleucine) were briefly presented the three-dimensional structure of AnSHMT (Figure 3).Figure 2

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