Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches

The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2‐fold improvement in kcat, 5.2‐fold lower Km and 16‐fold improvement in catalytic efficiency for D‐tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased kcat for D‐tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher kcat and Km value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D‐tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9‐fold) improvement in kcat and a 2.4‐fold increase in Km compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the Km up to 2.6‐fold for D‐tyrosine but one variant 145_V153A increased the Km 2.4‐fold compared to the WT. Homology based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an α‐helix providing one of the conserved histidine residues of the active site. The kcat and Km values for L‐tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1‐fold high catalytic efficiency compared to the WT which is a 7.6‐fold lower improvement compared to D‐tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D‐tyrosine:D‐DOPA and a 1.4‐fold higher L‐tyrosine:L‐DOPA activity ratio compared to the WT. Biotechnol. Bioeng. 2013; 110: 1849–1857. © 2013 Wiley Periodicals, Inc. Bacterial tyrosinases generally exhibit poor activity towards D‐tyrosine in comparison to L‐tyrosine. Through in vitro mutagenesis Ralstonia solanacearum tyrosinase variants with between 2.8‐ and 16‐fold higher catalytic efficiency for D‐tyrosine were obtained. Amino acid changes occurred outside the active site with T183I and N322S improving affinity while V153A improved catalytic activity but decreased affinity of the enzyme for D‐tyrosine. Engineered tyrosinases contain amino acid changes affecting D‐tyrosine more than L‐tyrosine oxidation.