Nakayama Norikazu, Matsubara Toshiyuki, Ohshiro Takashi, Moroto Yuko, Kawata Yasushi, Koizumi Kenichi, Hirakawa Yasuto, Suzuki Masanori, Maruhashi Kenji, Izumi Yoshikazu, Kurane Ryuichiro
Tsukuba Branch of Bio-Refining Process Laboratory, Advanced Technology and Research Institute, Petroleum Energy Center, 1-1 Higashi, Tsukuba, Japan.
Biochim Biophys Acta. 2002 Jul 29;1598(1-2):122-30. doi: 10.1016/s0167-4838(02)00365-5.
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.
二苯并噻吩（DBT）是石油中有机硫化合物的模型，经微生物脱硫生成2-羟基联苯（2-HBP），DBT脱硫需要基因操纵子dszABC。微生物DBT脱硫的最后一步是由DszB催化2'-羟基联苯-2-亚磺酸盐（HBPSi）转化为2-HBP。在本研究中，通过与伴侣蛋白基因groEL/groES共表达，在25℃下使DBT脱硫细菌红平红球菌KA2-5-1的DszB在大肠杆菌中过量表达。重组DszB被纯化至同质并进行了表征。DszB活性的最佳温度和pH分别为35℃和约7.5。HBPSi的K(m)和k(cat)值分别为8.2 microM和0.123.s(-1)。DszB只有一个半胱氨酸残基，当该半胱氨酸残基变为丝氨酸残基时，突变酶完全失去活性。该结果与使用抑制剂的实验表明，半胱氨酸残基有助于酶活性。DszB也受到反应产物2-HBP（K(i)=0.25 mM）及其衍生物的抑制，但不受另一种反应产物亚硫酸盐的抑制。该酶表现出狭窄的底物特异性：在所测试的芳香族和脂肪族亚磺酸盐或磺酸盐中，除HBPSi外只有2-苯基苯亚磺酸盐可作为底物。DszB被认为是一种新型酶（HBPSi脱亚磺酰酶）在于它可以在不借助任何其他蛋白质成分和辅酶的情况下特异性地裂解HBPSi的碳硫键，生成2-HBP和亚硫酸根离子。
