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Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches.
Molloy, Susan; Nikodinovic-Runic, Jasmina; Martin, Leona B; Hartmann, Hermann; Solano, Francisco; Decker, Heinz; O'Connor, Kevin E.
Afiliação
  • Molloy S; School of Biomolecular and Biomedical Sciences, Ardmore House, University College Dublin, Belfield, Dublin 4, Ireland.
Biotechnol Bioeng ; 110(7): 1849-57, 2013 Jul.
Article em En | MEDLINE | ID: mdl-23381872
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.
Assuntos

Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Tirosina / Engenharia de Proteínas / Monofenol Mono-Oxigenase / Ralstonia solanacearum Tipo de estudo: Clinical_trials Idioma: En Revista: Biotechnol Bioeng Ano de publicação: 2013 Tipo de documento: Article País de afiliação: Irlanda

Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Tirosina / Engenharia de Proteínas / Monofenol Mono-Oxigenase / Ralstonia solanacearum Tipo de estudo: Clinical_trials Idioma: En Revista: Biotechnol Bioeng Ano de publicação: 2013 Tipo de documento: Article País de afiliação: Irlanda