Elucidation of properties of novel enzymes catalyzing the formation and the cleavage of carbon-sulfur bond in microooganisms

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The research results of this study are summarized as follows.(1) Studies on biotin synthase(1-1) Biotin sythase (BioB), the enzyme responsible for the last step of biotin biosynthesis by microorganisms, is known to supply a sulfur atom in the Fe-S cluster of the enzyme. It was purified with a high yield and with a high purity from cell-free extract of a bioB transformant of Bacillus sphaericus which could overproduce BioB.Using the purified BioB enzyme preparation, biotin analogs, thiophene valeric acid (TVA) and acidomycin (ACM), were found to completely inhibit biotin synthase reaction at a concentration of 20 mM and 10 mM, respectively.(1-2) Stimulatory factors essential for biotin synthase reactions were found. Through ammonium sulfate fractionation and column chromatographies of cell-free extract of a wild type strain of B.sphaericus, a low-molecular weight fraction (LF), which was heat-stable and passed through ultramembrane of cutoff MW=10,000, and a high molecular weight fraction (HF), which was heat-lable and did not pass through the membrane, were obtained. Neither of LF and HF alone showed stimulatory activity toward biotin synthase reaction, but activity was obtained when both of them were added together in the reaction mixutre of biotin synthase. Since more than two stimulatory proteins may exist in the HF, further purification of HF was considered to be necessary(2) Studies on the purification of enzymes of dibenzothiophene (DBT) desulfurization metabolism(2-1) DBT monoxygenase (DszC) and DBT sulfone monooxygenase (DszA) were purified from a DBT desulfurizing Rhodococcus erythropolis D-1 and their reactions were found to essentially require reductase. In addition, DszC and DszA were successfully crystallized.(2-2) Sulfinase (DszB) which catalyzes the last step of DBT desulfurization metabolism, was also purified to a high degree from another DBT desulfurizing Rhodococcus erythropolis KA2-5-1 by various column chromatoraphies.