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

阐明微生物中催化碳硫键形成和断裂的新型酶的特性

基本信息

批准号：
11660091
负责人：
IZUMI Yoshikazu
金额：
$ 1.98万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660091/
关键词：
biotin BioB bioB dibenzothiophen (DBT)DszC,A フラビンレダクターゼ DszA ビオチン BioB bioB

项目摘要

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.

本研究的研究成果概括如下： (1)生物素合酶的研究(1-1)生物素合酶(BioB)是微生物生物合成生物素最后一步的酶，已知在生物素合成酶中提供硫原子。酶的 Fe-S 簇。从可过量生产BioB的球形芽孢杆菌bioB转化体的无细胞提取物中以高产率和高纯度纯化。使用纯化的BioB酶制剂、生物素类似物、噻吩戊酸(TVA)和酸霉素(ACM) ，被发现在浓度分别为 20 mM 和 10 mM 时完全抑制生物素合酶反应。(1-2) 生物素合酶反应必需的刺激因子发现了生物素合酶反应。通过对球形芽孢杆菌野生型菌株的无细胞提取物进行硫酸铵分级分离和柱层析，得到低分子量组分（LF），该组分对热稳定并能通过截留分子量=10,000的超膜，并且具有高分子量组分（LF）。获得了不耐热且不穿过膜的分子量级分（HF）。单独的LF和HF均未表现出对生物素合酶反应的刺激活性，但当将两者一起添加到生物素合酶的反应混合物中时获得活性。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)DBT单加氧酶(DszC)和DBT砜单加氧酶 (DszA) 从 DBT 脱硫红平红球菌 D-1 中纯化，发现它们的反应基本上需要还原酶。此外，DszC和DszA也成功结晶。(2-2)催化DBT脱硫代谢最后一步的磺化酶(DszB)也通过各种柱从另一种DBT脱硫红平红球菌KA2-5-1中得到高度纯化。色谱法。