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

本研究的研究结果总结如下：(1)生物素合成酶的研究（1-1）生物素合成酶（biotin sythase, BioB）是微生物合成生物素的最后一步酶，已知在酶的Fe-S簇中提供一个硫原子。从能过量产生bioB的球形芽孢杆菌（Bacillus sphaericus）的一种bioB转化菌的无细胞提取物中纯化出高产、高纯度的菌株。利用纯化的BioB酶制剂，发现生物素类似物、噻吩戊酸（TVA）和酸霉素（ACM）分别在20 mM和10 mM浓度下完全抑制生物素合成酶反应。（1-2）发现了生物素合成酶反应必需的刺激因子。通过硫酸铵分馏法和柱层析对一种野生型球形芽孢杆菌的无细胞提取物进行分离，得到了热稳定的低分子量组分（LF）和热稳定的高分子量组分（HF），并通过了切断MW=10,000的超膜。LF和HF单独对生物素合成酶反应均不具有刺激活性，但在生物素合成酶反应混合物中加入LF和HF均具有刺激活性。由于HF中可能存在两种以上的刺激蛋白，因此认为有必要进一步纯化HF(2)二苯并噻吩（DBT）脱硫代谢酶的纯化研究（2-1）从一株DBT脱硫红红红球菌D-1中纯化出DBT单加氧酶（DszC）和DBT砜单加氧酶（DszA），发现它们的反应基本需要还原酶。DszC和DszA均成功结晶。（2-2）催化DBT脱硫代谢最后一步的亚砜酶（Sulfinase, DszB）也通过各种柱层析从另一种DBT脱硫红红球菌KA2-5-1中得到了高度纯化。

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.