The effect of mutations near the T1 copper site on the biochemical characteristics of the small laccase from Streptomyces coelicolor A3(2)

A Prins; L Kleinsmidt; N Khan; B Kirby; T Kudanga; J Vollmer; J Pleiss; S Burton; M Le Roes-Hill

doi:10.1016/j.enzmictec.2014.10.003

The effect of mutations near the T1 copper site on the biochemical characteristics of the small laccase from Streptomyces coelicolor A3(2)

Enzyme Microb Technol. 2015 Jan:68:23-32. doi: 10.1016/j.enzmictec.2014.10.003. Epub 2014 Oct 23.

Authors

A Prins¹, L Kleinsmidt², N Khan³, B Kirby⁴, T Kudanga⁵, J Vollmer⁶, J Pleiss⁷, S Burton⁸, M Le Roes-Hill⁹

Affiliations

¹ Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa; Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, PO Box X17 Bellville, 7535, South Africa. Electronic address: alaric.prins@gmail.com.
² Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa. Electronic address: lkleins@gmail.com.
³ Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa. Electronic address: nuraankhan@gmail.com.
⁴ Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, PO Box X17 Bellville, 7535, South Africa. Electronic address: bkirby@uwc.ac.za.
⁵ Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa; Department of Biotechnology and Food Technology, Durban University of Technology, P.O. BOX 1334, Durban, 4000, South Africa. Electronic address: tukayik@dut.ac.za.
⁶ Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, Stuttgart, D-70569, Germany. Electronic address: jannikvollmer@gmail.com.
⁷ Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, Stuttgart, D-70569, Germany. Electronic address: itbjpl@itb.uni-stuttgart.de.
⁸ University of Pretoria, Private Bag X20, Hatfield, Pretoria, 0028, South Africa. Electronic address: stephanie.burton@up.ac.za.
⁹ Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa. Electronic address: LeroesM@cput.ac.za.

PMID: 25435502
DOI: 10.1016/j.enzmictec.2014.10.003

Abstract

Bacterial laccases show low activities but can be of biotechnological interest due to industrially suitable characteristics such as thermostability and tolerance to alkaline pH. In this study, three separate mutations (M298F, V290N and V290A) were introduced at or near the T1 copper site of the small laccase (SLAC) from Streptomyces coelicolor A3(2) and biochemical properties were assessed in comparison with the native enzyme. The mutation, V290N showed approximately double the activity of SLAC when ABTS was used as substrate while the specific activity of SLAC-M298F was 4-5 times higher than that of SLAC when the assays were performed at ≥70°C. There was no significant difference in activity with 2,6-dimethoxyphenol (2,6-DMP); however, there was a significant shift in the optimal pH from pH 9.5 (SLAC) to 7.5 (SLAC-V290N). Optimal temperature for activity was not significantly altered but thermostability was reduced in all three mutants. The substrate range of the mutant variants remained largely unchanged, with the exception of SLAC-M298F which was unable to oxidise veratryl alcohol. Interestingly, the "typical" laccase inhibitor, sodium azide, had no significant inhibitory effect on the activity of SLAC-M298F, which also exhibited increased resistance to inhibition by sulfhydryl compounds. SLAC-V290N showed higher catalytic efficiency for 2,6-DMP (kcat/Km=2.226mM(-1)s(-1)) and ABTS (kcat/Km=1.874mM(-1)s(-1)) compared to SLAC (kcat/Km=1.615mM(-1)s(-1) for 2,6-DMP and kcat/Km=1.611mM(-1)s(-1) for ABTS). This study has shown that three ligands that are closely associated with the T1 copper in SLAC play a key role in maintaining enzymatic activity. Whilst the introduction of mutations at these sites negated favourable characteristics such as thermostability, several favourable effects were observed. This study has also extended the knowledge base on the biochemical characteristics of SLAC, and its suitability as a template for engineering with the aim of widening its potential range of industrial applications.

Keywords: Biochemical characteristics; Small laccase; T1 copper, Mutation.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Substitution*
Bacterial Proteins / antagonists & inhibitors
Bacterial Proteins / chemistry
Bacterial Proteins / genetics*
Bacterial Proteins / metabolism
Benzothiazoles / metabolism
Copper
Hydrogen-Ion Concentration
Laccase / antagonists & inhibitors
Laccase / chemistry
Laccase / genetics*
Laccase / metabolism
Ligands
Molecular Structure
Mutagenesis, Site-Directed
Mutation, Missense*
Point Mutation*
Protein Stability
Pyrogallol / analogs & derivatives
Pyrogallol / metabolism
Sequence Alignment
Sequence Homology, Nucleic Acid
Sodium Azide / pharmacology
Streptomyces coelicolor / enzymology*
Streptomyces coelicolor / genetics
Substrate Specificity
Sulfhydryl Compounds / pharmacology
Sulfonic Acids / metabolism
Temperature

Substances

Bacterial Proteins
Benzothiazoles
Ligands
Sulfhydryl Compounds
Sulfonic Acids
Pyrogallol
2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid
pyrogallol 1,3-dimethyl ether
Copper
Sodium Azide
Laccase