臨床検査への応用を目指した微生物由来ヒドロキシステロイドデヒドロゲナーゼの分子学及び酵素学的研究

概要

論文の詳細を見る
In clinical diagnostics, 3α-hydroxysteroid dehydrogenases have been used to measure the total bile acids (TBA) in serum. Dual-nucleotide cofactor-specific 3α-HSD from Pseudomonas sp. B-0831, a member of the SDR superfamily, catalyzed the oxidation of the antibiotic fusidic acid efficiently with the lowest K_m value among various 3α-hydroxysteroids. The enzyme showed the utility of cofactor analogues, such as thio-NAD^+, 3-acetylpyridine adenine dinucleotide (APAD^+) and nicotinamide hypoxanthine dinucleotide (deamino-NAD^+), and could catalyze the carbonyl reduction of non-steroid substrates, and thus can be named 3α-HSD/CR. Transient-phase kinetic studies using the fluorescence stopped-flow method were conducted with the 3α-HSD, for the first time as prokaryotic 3α-HSD, to characterize the binding mechanism of the nucleotide cofactor. The binding of the oxidized cofactors, NAD^+ and NADP^+, agreed well with a one-step mechanism, while that of a reduced cofactor, NADH, showed a two-step mechanism. These results are different from that of 3α-HSD from rat liver which belongs to the AKR superfamily. A simple kinetic model of a highly sensitive enzymatic cycling assay method for TBA, previously developed by utilizing reversible catalytic function of the 3α-HSD in the presence of different kinds of oxidized and reduced cofactors, thio-NAD^+ and NADH, was proposed and confirmed by the experimental results.
2005-03-05