title: 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.
authors: School of Biomolecular and Biomedical Sciences, Ardmore House, University College Dublin, Belfield, Dublin 4, Ireland.
authors: Susan
authors: S
authors: Molloy
authors: Jasmina
authors: J
authors: Nikodinovic-Runic
authors: Leona B
authors: LB
authors: Martin
authors: Hermann
authors: H
authors: Hartmann
authors: Francisco
authors: F
authors: Solano
authors: Heinz
authors: H
authors: Decker
authors: Kevin E
authors: KE
authors: O'Connor
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011487:Protein Conformation; D015202:Protein Engineering; D043368:Ralstonia solanacearum / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D014442:Monophenol Monooxygenase