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

Abstract

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.

Figure 1

Substrate selectivity of small laccase (SLAC). Substrate oxidation by purified wild-type SLAC (dark grey bars) and variant S292A (light grey bars) on various substrates. Wild-type SLAC and the S292A variant were prepared using microaerobic growth conditions. Substrate depletion (μM) was measured after 20 min at 60°C and the optimal pH for the reaction. Initial substrate concentrations were 1000 μM. n = 3; errors indicate standard deviation.

Figure 2

Kinetic parameters of wild-type small laccase (SLAC) and the S292A variant enzyme. Kinetic parameters were determined for wild-type (wt) SLAC on ABTS, 2,6-DMP, quercetin, morin and myricetin. Kinetic parameters were also determined for S292A on ABTS, 2,6-DMP, and morin, but could not be obtained for quercetin and myricetin since the apparent K_m of the enzyme for these substrates exceeded substrate solubility. Lighter shaded regions in quercetin, morin and myricetin molecules indicate identical chemical structures. n = 3; errors indicate standard deviation.

Figure 3

Percent activity of small laccase (SLAC) variants on ABTS relative to the wild-type enzyme.A. amino acid substitutions at positions that coordinate T1 and T2/T3 copper ions.B. amino acid substitutions at positions predicted to participate in substrate binding. The comparatively high activity of Y230A and Y229A mutants likely results from higher copper incorporation. Reactions (200 μl) contained 2 μg of enzyme prepared from standard cultivation conditions, 1 mM ABTS and 50 mM sodium acetate (pH 4.0). n = 3; errors indicate standard deviation.