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

6533b85afe1ef96bd12b8c01

RESEARCH PRODUCT

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

Kevin E. O’connor Susan Molloy Francisco Solano Jasmina Nikodinovic-runic Heinz Decker Leona B. Martin Hermann Hartmann

subject

DNA Bacterial Protein Conformation Sequence analysis Tyrosinase homology modeling Molecular Sequence Data Mutation Missense random mutagenesis Bioengineering tyrosinase Protein Engineering 010402 general chemistry 01 natural sciences Applied Microbiology and Biotechnology enzyme catalysis 03 medical and health sciences site specific mutagenesis Missense mutation Site-directed mutagenesis Histidine 030304 developmental biology 0303 health sciences Ralstonia solanacearum biology Monophenol Monooxygenase Wild type Active site Sequence Analysis DNA biology.organism_classification Molecular biology Recombinant Proteins 0104 chemical sciences Kinetics Mutagenesis Ralstonia solanacearum biology.protein Tyrosine D-tyrosine Mutant Proteins Biotechnology

description

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 -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. This is the peer-reviewed version of the following article: Molloy, S.; Nikodinović-Runić, J.; Martin, L. B.; Hartmann, H.; Solano, F.; Decker, H.; O’Connor, K. E. Engineering of a Bacterial Tyrosinase for Improved Catalytic Efficiency towards D-Tyrosine Using Random and Site Directed Mutagenesis Approaches. Biotechnology and Bioengineering 2013, 110 (7), 1849–1857. [https://doi.org/10.1002/bit.24859] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3492]

year	journal	country	edition	language
2013-02-22

10.1002/bit.24859 http://cherry.chem.bg.ac.rs/handle/123456789/1359