Funding Information Funding for this research was provided by the Government of Ontario for the project ‘FFABnet: Functionalized Fibre and Biochemicals’ (ORF-RE-05-005), and the Natural Sciences and Engineering Research Council of Canada.
Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site-directed mutagenesis
Article first published online: 1 JUL 2013
© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 6, Issue 5, pages 588–597, September 2013
How to Cite
Sherif, M., Waung, D., Korbeci, B., Mavisakalyan, V., Flick, R., Brown, G., Abou-Zaid, M., Yakunin, A. F. and Master, E. R. (2013), Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site-directed mutagenesis. Microbial Biotechnology, 6: 588–597. doi: 10.1111/1751-7915.12068
- Issue published online: 14 AUG 2013
- Article first published online: 1 JUL 2013
- Manuscript Accepted: 1 MAY 2013
- Manuscript Revised: 29 APR 2013
- Manuscript Received: 30 NOV 2012
- Government of Ontario for the project ‘FFABnet: Functionalized Fibre and Biochemicals’. Grant Number: ORF-RE-05-005
- Natural Sciences and Engineering Research Council of Canada
Multicopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60°C were 8.1 (± 0.8) s−1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half-life of over 10 h at 80°C and over 7 h at 90°C. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates.