Title
Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus
Author
Gaobing Wu, Tao Zhan, Yiming Guo, Ashok Kumar, Ziduo Liu
Year
2016
Journal
Electronic Journal of Biotechnology
Abstract
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 new glycine 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. 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. 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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry