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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 22, No. 1, 2016, pp. 26-30
Bioline Code: ej16032
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 22, No. 1, 2016, pp. 26-30

 en Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus check for this species in other resources
Wu, Gaobing; Zhan, Tao; Guo, Yiming; Kumar, Ashok & Liu, Ziduo

Abstract

Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study,we have characterized a newglycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking.
Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between β-18 and α-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme.
Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure–function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.

Keywords
Bacillus cereus; Error-prone PCR; Glycine oxidase; Site-directed mutagenesis; Substrate affinity

 
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