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

Abstract

tBacterial laccases show low activities but can be of biotechnological interest due to industrially suit-able characteristics such as thermostability and tolerance to alkaline pH. In this study, three separatemutations (M298F, V290N and V290A) were introduced at or near the T1 copper site of the small lac-case (SLAC) from Streptomyces coelicolor A3(2) and biochemical properties were assessed in comparisonwith the native enzyme. The mutation, V290N showed approximately double the activity of SLAC whenABTS was used as substrate while the specific activity of SLAC-M298F was 4–5 times higher than that ofSLAC 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 thermostabilitywas 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 “typ-ical” 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 showedhigher catalytic efficiency for 2,6-DMP (kcat/Km= 2.226 mM−1s−1) and ABTS (kcat/Km= 1.874 mM−1s−1)compared to SLAC (kcat/Km= 1.615 mM−1s−1for 2,6-DMP and kcat/Km= 1.611 mM−1s−1for ABTS). Thisstudy has shown that three ligands that are closely associated with the T1 copper in SLAC play a key rolein maintaining enzymatic activity. Whilst the introduction of mutations at these sites negated favourablecharacteristics such as thermostability, several favourable effects were observed. This study has alsoextended the knowledge base on the biochemical characteristics of SLAC, and its suitability as a templatefor engineering with the aim of widening its potential range of industrial applications.