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FEBS Open Bio. 2019 May;9(5):986-995. doi: 10.1002/2211-5463.12633. Epub 2019 Apr 9.

Axial bonds at the T1 Cu site of Thermus thermophilus SG0.5JP17-16 laccase influence enzymatic properties.

Zhu Y1,2, Zhang Y1,2, Zhan J1,2, Lin Y1,2, Yang X1,2.

Author information

1
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
2
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.

Abstract

Laccase is a multi-copper oxidase which oxidizes substrate at the type 1 copper site, simultaneously coupling the reduction of dioxygen to water at the trinuclear copper center. In this study, we used site-directed mutagenesis to study the effect of axial bonds between the metal and amino acid residue side chains in lacTT. Our kinetic and spectral data showed that the replacement of the axial residue with non-coordinating residues resulted in higher efficiency (kcat /Km ) and a lower Cu2+ population at the type 1 copper site, while substitution with strongly coordinating residues resulted in lower efficiency and a higher Cu2+ population, as compared with the wild-type. The redox potentials of mutants with hydrophobic axial residues (Ala and Phe) were higher than that of the wild-type. In conclusion, these insights into the catalytic mechanism of laccase may be of use in protein engineering to fine-tune its enzymatic properties for industrial application.

KEYWORDS:

axial bond; copper; kinetic analysis; laccase; redox potential; site-directed mutation

PMID:
30964606
PMCID:
PMC6487685
DOI:
10.1002/2211-5463.12633
[Indexed for MEDLINE]
Free PMC Article

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