Construction and Characterization of Composite Biocatalysts

分析了铁硫簇合物的生物合成机理，认为铁硫簇合物可作为复合生物催化剂的组分。我们还开发了新的方法，通过使用新型复合生物催化剂的手性化合物的生产。此外，还阐明了以磷酸吡哆醛为辅酶的氨基酸消旋酶等几种复合生物催化剂的反应机理.铁硫簇合物的硫原子由L-半胱氨酸通过半胱氨酸脱硫酶催化的硫消除反应提供。我们研究了铁-硫簇组装的详细机制，涉及以下半胱氨酸脱硫酶：来自大肠杆菌的IscS和SufS以及来自集胞藻属PCC 6803的SsCsd 3。分析了这些半胱氨酸脱硫酶与作为铁硫簇组装支架蛋白的蛋白质之间的相互作用.我们分离并鉴定了一种新的NADPH酶，N-甲基-L-氨基酸脱氢酶。从恶臭假单胞菌ATCC 12633中克隆了编码该酶的基因，并构建了其过表达系统。利用该酶，我们开发了一种由α-酮酸和甲胺制备手性N-甲基-L-氨基酸的酶法.我们分离并鉴定了一种新的NADPH酶，其催化卤代不饱和化合物中碳-碳双键的不对称还原。我们开发了一种酶促方法，其中（S）-2-CPA，这是作为一个构件块用于合成芳氧基苯氧基丙酸除草剂，是通过不对称还原的碳-碳双键在2-氯丙烯酸酯。

We analyzed the mechanism of biosynthesis of iron-sulfur clusters, which are useful as components of composite biocatalysts. We also developed novel processes for the production of chiral compounds by the use of novel composite biocatalysts. In addition, reaction mechanisms of several composite biocatalysts such as amino acid racemases which require pyridoxal phosphate as a coenzyme were clarified.1. Sulfur atoms of iron-sulfur clusters are supplied from L-cysteine by the sulfur-elimination reaction catalyzed by cysteine desulfurases. We studied the detailed mechanisms of iron-sulfur cluster assembly involving the following cysteine desulfurases : IscS and SufS from Escherichia coli and SsCsd3 from Synechocystis sp. PCC6803. Interactions between these cysteine desulfurases and proteins functioning as scaffold proteins for the assembly of iron-sulfur clusters were analyzed.2. We isolated and characterized a novel NADPH enzyme, N-methyl-L-amino acid dehydrogenase. The gene coding for this enzyme was cloned from Pseudomonas putida ATCC12633, and its overexpression system was constructed. Using this enzyme, we developed an enzymatic process in which chiral N-methyl-L-amino acids are produced from α-keto acids and methylamine.3. We isolated and characterized a novel NADPH enzyme that ctalyzes the asymmetric reduction of a carbon-carbon double bond in halogenated unsaturated compounds. We developed an enzymatic process in which (S)-2-CPA, which is used as a building block for the synthesis of aryloxyphenoxypropionic acid herbicide, is produced by asymmetric reduction of a carbon-carbon double bond in 2-chloroacrylate.