Purification and Characterization of Recombinant 2‐(2′‐hydroxyphenyl)benzene sulfinate desulfinase from Nocardia asteroides strain sp. A3H1

Abstract

Nocardia asteroides strain sp. A3H1 is a bacterial strain that can metabolize dibenzothiophene (DBT), a major contaminant in diesel fuel, via a sulfur specific oxidative desulfurization pathway. The enzyme 2-(2′-hydroxyphenyl)benzene sulfinate desulfinase (DszB) catalyzes the final rate-limiting step in the desulfurization pathway. Previous chemical modification studies on the native DszB enzyme from Rhodococcus erythropolis strain sp. IGTS8 support the presence of tyrosine, tryptophan and cysteine residues in the active site. Furthermore, amino acid sequence analysis of the DszB enzyme shows there is only one cysteine residue and only one conserved tyrosine residue. The wild-type and mutant recombinant A3H1 DszB enzymes were purified, characterized and used to investigate the mechanism of this enzyme. Fluorometric activity assays were conducted to determine optimal conditions (pH, temperature, etc.) for stability and use of the DszB enzyme. Amino acids Cys27 and Tyr24 that are proposed to be important in the active site of the enzyme, were mutated via site-directed mutagenesis. The mutant enzymes’ characteristics were compared to those of the recombinant wild-type enzyme. Thus, the effects of these point mutations in the DszB enzyme contribute to our understanding of the mechanism for the removal of sulfur from DBT.