Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus |
Wu, Gaobing; Zhan, Tao; Guo, Yiming; Kumar, Ashok & Liu, Ziduo
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.
Bacillus cereus; Error-prone PCR; Glycine oxidase; Site-directed mutagenesis; Substrate affinity