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

Abstract

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 k(cat), 5.2-fold lower K(m) 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 k(cat) for D-tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher k(cat) and K(m) 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 k(cat) and a 2.4-fold increase in K(m) compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the K(m) up to 2.6-fold for D-tyrosine but one variant 145_V153A increased the K(m) 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 k(cat) and K(m) 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.

Total Synthesis, Stereochemical Assignment, and Divergent Enantioselective Enzymatic Recognition of Larreatricin

“…[29] Another bacterial tyrosinase from Ralstonias olanacearum showed am edium enantiopreference for l-tyrosine (4.7 times faster than on the R-enantiomer) which could be reduced to almost no selectivity (0.98, that is, av ery slight preference for the Renantiomer) by mutation of one amino acid far from the active site. [30] Enantioselectivities similar to the values presented here for larreatricin were observed for as ecreted fungal tyrosinase from Trichoderma reesei that reacted1 4.3 times faster on 2.5 mm l-tyrosine than on its R-enantiomer (based on specific activitiesb ecause no kinetic characterization was given). [31] The higheste nantioselectivity reported for aP PO comes from tyrosinase produced by the bacterium Streptomyces sp.…”

Microbial Tyrosinases: Promising Enzymes for Pharmaceutical, Food Bioprocessing, and Environmental Industry

“…In fact, almost all studies on tyrosinase inhibition conducted so far have used mushroom tyrosinase because the enzyme is commercially available [11]. It was investigated by Molloy et al [12] recently that bacterial tyrosinase from Ralstonia solanacearum can be modified through engineering which in turn is used for the improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis. Mushrooms are considered a cheap source of the tyrosinase.…”

Highly Sensitive Phenol Biosensor Utilizing Selected Bacillus Biofilm Through an Electrochemical Method

“…A solution for that problem is using selected bacteria as bioreceptors as they contain natural enzymatic pathways [12]. Several bacteria that produce tyrosinase, such as Bacillus thuringiensis, Pseudomonas putida [13][14][15], and Ralstonia solanacearum [16][17], have been reported. However, the Bacillus genus is resistant to extreme conditions while producing tyrosinase enzyme [18].…”