Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus

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 new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against a...

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Autores principales:	Wu,Gaobing, Zhan,Tao, Guo,Yiming, Kumar,Ashok, Liu,Ziduo
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2016
Materias:	Bacillus cereus Error-prone PCR Glycine oxidase Site-directed mutagenesis Substrate affinity
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400004
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spelling	oai:scielo:S0717-345820160004000042016-09-13Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereusWu,GaobingZhan,TaoGuo,YimingKumar,AshokLiu,Ziduo Bacillus cereus Error-prone PCR Glycine oxidase Site-directed mutagenesis Substrate affinity 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 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. 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 \|J-18 and a-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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.19 n.4 20162016-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400004en10.1016/j.ejbt.2016.02.009
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Bacillus cereus Error-prone PCR Glycine oxidase Site-directed mutagenesis Substrate affinity
spellingShingle	Bacillus cereus Error-prone PCR Glycine oxidase Site-directed mutagenesis Substrate affinity Wu,Gaobing Zhan,Tao Guo,Yiming Kumar,Ashok Liu,Ziduo Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus
description	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 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. 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 \|J-18 and a-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.
author	Wu,Gaobing Zhan,Tao Guo,Yiming Kumar,Ashok Liu,Ziduo
author_facet	Wu,Gaobing Zhan,Tao Guo,Yiming Kumar,Ashok Liu,Ziduo
author_sort	Wu,Gaobing
title	Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus
title_short	Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus
title_full	Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus
title_fullStr	Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus
title_full_unstemmed	Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus
title_sort	asn336 is involved in the substrate affinity of glycine oxidase from bacillus cereus
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2016
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400004
work_keys_str_mv	AT wugaobing asn336isinvolvedinthesubstrateaffinityofglycineoxidasefrombacilluscereus AT zhantao asn336isinvolvedinthesubstrateaffinityofglycineoxidasefrombacilluscereus AT guoyiming asn336isinvolvedinthesubstrateaffinityofglycineoxidasefrombacilluscereus AT kumarashok asn336isinvolvedinthesubstrateaffinityofglycineoxidasefrombacilluscereus AT liuziduo asn336isinvolvedinthesubstrateaffinityofglycineoxidasefrombacilluscereus
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Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus

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