Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

Abstract

SummaryMulticopper 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.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.

Structural and functional characterization of two-domain laccase from Streptomyces viridochromogenes

“…Lack of tyrosine oxidation and a very weak activity against L-DOPA shows SvSL not to participate in the metabolism of tyrosine. A similar situation was observed for other 2DLac from Streptomyces, which also oxidize phenolic compounds less efficiently than electron donors [20,21,51]. SvSL did not possess any ferroxidase activity, so its role is not related to the metabolism of iron in the cell.…”

Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades

“…[73] The enzyme concentrations were chosen on the basis of previous experience to achieve maximum oxidation of the galactosyl residues in each substrate. [75,76] w-TAr eactions and activity assays After verifying that the oxidation reaction had reached maximum conversion (evaluated by TLC, data not shown), the activity of the purified Cvi-w-TAt owards oxidized carbohydrates was measured by chromogenic detection of acetophenone from 1-phenylethylamine (1-PEA). Conversion of each oxidation reaction was eval-uated by TLC (data not shown) on Macherey-Nagel precoated silica gel plates (TLC Silica gel 60 F254).…”

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

“…In contrast, the affinity for phenolic substrates was greatly improved whilst 10 the maximum reaction rate remained largely unchanged. When comparing the catalytic efficiency of SLAC to the work performed by Sherif et al (2013), the SLAC used in this study exhibited a lower k cat /K m value (1.611 mM -1 s -1 ) compared to 10 mM -1 s -1…”