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Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids.

ABSTRACT: Unnatural amino acids (UAAs) are chiral amines with high application potential in drug discovery and synthesis of other valuable chemicals. Biocatalysis offers the possibility to synthesise novel optically pure UAAs with different physical and chemical properties. While the biocatalytic potential of transaminases in the synthesis of UAAs has been demonstrated, there is still a need to improve the activity with non-native substrates and to understand which amino acids residues are important for activity with these UAAs. Using a rational design approach, six variants of Chromobacterium violaceum DSM30191 transaminase (CV_TA) carrying a single and one variant carrying two substitutions were generated. Among the variants with a single substitution, CV_Y168F showed a 2 to 2.6-fold increased affinity for 2-oxooctanoic acid (2-OOA) and 3-oxobutyric acid (3-OBA) methyl ester used to synthesise an ?- and ?-UAA. Analysis of the first half of the transaminase reaction showed no change in the activity with the donor (S)-1-phenylethylamine. The combination of W60C and Y168F substitutions improved the CV_TA affinity for 2-OOA 10-fold compared to the wild type. Other substitutions showed no change, or reduced activity with the tested substrates. Our findings provide structural information on CV_TA and demonstrate the potential of rational design for biosynthesis of UAAs.

SUBMITTER: Almahboub SA

PROVIDER: S-EPMC6255834 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids.

Almahboub Sarah A SA Narancic Tanja T Fayne Darren D O'Connor Kevin E KE

Scientific reports 20181126 1

Unnatural amino acids (UAAs) are chiral amines with high application potential in drug discovery and synthesis of other valuable chemicals. Biocatalysis offers the possibility to synthesise novel optically pure UAAs with different physical and chemical properties. While the biocatalytic potential of transaminases in the synthesis of UAAs has been demonstrated, there is still a need to improve the activity with non-native substrates and to understand which amino acids residues are important for a ...[more]

PMID: 30478262

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Project description:The antiviral mechanism of sulfated polysaccharides is supposed to prevent virus entry, which is mediated by the interactions of anionic charges on sulfated polysaccharides with positively charged domains of viral envelope glycoproteins, leading to shielding of the functional domain involved in virus attachment to cell surface receptors. But, few direct evidences were reported. In the previous study, we found that sulfated Chuanmingshen violaceum polysaccharides (sCVPS) possessed remarkable inhibitory effect against Newcastle disease virus (NDV) through inhibition of NDV attachment to host cells. Whether sCVPS bound to hemagglutinin-neuraminidase (HN) leading to inhibition of NDV attachment needs to be further clarified. The present study conducted site-directed mutagenesis of possible positively charged residues of HN, and found that mutants R197G, H199G, R363G, and R523G could significantly decrease the inhibitory effects of sCVPS on receptor binding ability through hemadsorption assay, especially R363G which suggested that binding to R363 is more effective to shield the sialic acid binding sites. Dual mutants (R363G/R197G, R363G/H199G and R363G/R523G) induced more decreased inhibitory effect of sCVPS than single mutants. The immunofluorescence study using FITC-labeled sCVPS found that the fluorescence intensity of mutants R363G and R363G/H199G were significantly decreased. The binding kinetics of sCVPS to HN measured by surface plasmon resonance indicated that sCVPS had a higher binding affinity for wild-type HN than mutants R363G and R363G/H199G. Plaque reduction study was performed using recombinant NDV with mutant HNR363G and HNR363G/H199G, which showed significantly decreased inhibitory effects of sCVPS against mutant NDV adsorption to BHK-21 cells. These results suggested that the residues R197, H199, R363, and R523 were the binding sites for sCVPS, especially R363 act as the main interaction site. The present study provided direct evidence for the theory that antiviral mechanism of sulfated polysaccharides attributed to anionic groups binding to the positively charged residues of viral proteins which led to the shielding of receptor binding sites.

Dataset Information

Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids.

Publications

Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids.

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