A novel enzyme, 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB), from a dibenzothiophene-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1: gene overexpression and enzyme characterization.
Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl (2-HBP), and the gene operon dszABC was required for DBT desulfurization. The final step in the microbial DBT desulfurization is the conversion of 2'-hydroxybiphenyl-2-sulfinate (HBPSi) to 2-HBP catalyzed by DszB. In this study, DszB of a DBT-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 was overproduced in Escherichia coli by coexpression with chaperonin genes, groEL/groES, at 25 degrees C. The recombinant DszB was purified to homogeneity and characterized. The optimal temperature and pH for DszB activity were 35 degrees C and about 7.5, respectively. The K(m) and k(cat) values for HBPSi were 8.2 microM and 0.123.s(-1), respectively. DszB has only one cysteine residue, and the mutant enzyme completely lost the activity when the cysteine residue was changed to a serine residue. This result together with experiments using inhibitors showed that the cysteine residue contributes to the enzyme activity. DszB was also inhibited by a reaction product, 2-HBP (K(i)=0.25 mM), and its derivatives, but not by the other reaction product, sulfite. The enzyme showed a narrow substrate specificity: only 2-phenylbenzene sulfinate except HBPSi served as a substrate among the aromatic and aliphatic sulfinates or sulfonates tested. DszB was thought to be a novel enzyme (HBPSi desulfinase) in that it could specifically cleave the carbon-sulfur bond of HBPSi to give 2-HBP and sulfite ion without the aid of any other proteinic components and coenzymes.
Nakayama
Norikazu
N
Tsukuba Branch of Bio-Refining Process Laboratory, Advanced Technology and Research Institute, Petroleum Energy Center, 1-1 Higashi, Tsukuba, Japan.
Matsubara
Toshiyuki
T
Ohshiro
Takashi
T
Moroto
Yuko
Y
Kawata
Yasushi
Y
Koizumi
Kenichi
K
Hirakawa
Yasuto
Y
Suzuki
Masanori
M
Maruhashi
Kenji
K
Izumi
Yoshikazu
Y
Kurane
Ryuichiro
R
D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology*; D001426:Bacterial Proteins
D017931:DNA Primers; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011994:Recombinant Proteins; C113832:2-(2-hydroxybiphenyl)-benzenesulfinate desulfinase; D001426:Bacterial Proteins