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Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D‐tyrosine using random and site directed mutagenesis approaches
Author(s) -
Molloy Susan,
NikodinovicRunic Jasmina,
Martin Leona B.,
Hartmann Hermann,
Solano Francisco,
Decker Heinz,
O'Connor Kevin E.
Publication year - 2013
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24859
Subject(s) - tyrosinase , tyrosine , histidine , mutagenesis , site directed mutagenesis , chemistry , mutation , microbiology and biotechnology , ralstonia solanacearum , homology modeling , biochemistry , gene , biology , enzyme , stereochemistry , genetics , mutant , bacteria
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. Biotechnol. Bioeng. 2013; 110: 1849–1857. © 2013 Wiley Periodicals, Inc.