RESUMO
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
Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Engenharia de Proteínas , Ralstonia solanacearum/enzimologia , Tirosina/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Cinética , Dados de Sequência Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica , Ralstonia solanacearum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNARESUMO
Two melanin-overproducing Pseudomonas putida F6 mutants were generated using transposon (Tn5) mutagenesis. Mutants were disrupted in a transcriptional regulator (TR) and a homogentisate 1,2-dioxygenase (HDO) gene. Colonies of mutant F6-TR overproduced a black pigment on solid medium. The same mutant (F6-TR) had a 3.7-fold higher tyrosinase activity compared with the wild-type strain when induced with ferulic acid. However in tyrosine uptake assays whole cells of the mutant strain F6-TR consumed eight times less tyrosine compared with the wild-type strain. Mutant F6-HDO produced a diffusible red pigment into the growth medium. Pigment production by mutant F6-HDO is sixfold higher than the wild-type strain. The biomass yield of mutant F6-HDO grown on tyrosine as the sole source of carbon and energy was 1.2-fold lower than the wild-type strain. While the growth of the wild-type strain was completely inhibited by 5 min of exposure to UV light (254 nm) both mutant strains showed survival rates >30%. Mutant F6-HDO was able to tolerate higher concentrations of hydrogen peroxide (H(2)O(2)) exhibiting 1.5 times smaller zones of inhibition at 10 mM H(2)O(2) compared with mutant F6-TR and the wild-type strain. The pigments produced by all strains were purified and confirmed to be melanins.
