来自嗜热绿菌的产生L-苏式二氢生物蝶呤的蝶啶还原酶。
Sepiapterin reductase producing L-threo-dihydrobiopterin from Chlorobium tepidum.
作者信息
Cho S H, Na J U, Youn H, Hwang C S, Lee C H, Kang S O
机构信息
Laboratory of Biophysics, Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, Republic of Korea.
出版信息
Biochem J. 1999 Jun 1;340 ( Pt 2)(Pt 2):497-503.
A novel type of NADPH-dependent sepiapterin reductase, which catalysed uniquely the reduction of sepiapterin to l-threo-dihydrobiopterin, was purified 533-fold from the cytosolic fraction of Chlorobium tepidum, with an overall yield of 3%. The native enzyme had a molecular mass of 55 kDa and SDS/PAGE revealed that the enzyme consists of two subunits with a molecular mass of 26 kDa. The enzyme was optimally active at pH8.8 and 50 degrees C. Apparent Km values for sepiapterin and NADPH were 21 and 6.2 microM, respectively, and the kcat value was 5.0 s-1. Diacetyl could also serve as a substrate, with a Km of 4.0 mM. The inhibitory effects of N-acetylserotonin, N-acetyldopamine and melatonin were very weak. The Ki value of N-acetyldopamine was measured as 400 microM. The N-terminal amino acid sequence was revealed as Met-Lys-His-Ile-Leu-Leu-Ile-Thr-Gly-Ala-Xaa-Lys - Lys - Ile - Xaa - Arg - Ala - Ile - Ala - Leu - Glu - Xaa - Ala - Arg - Xaa-Xaa-Xaa-His-His-His-, which shared relatively high sequence similarity with other sepiapterin reductases.
从嗜热绿菌的胞质部分中纯化出一种新型的依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的蝶啶还原酶，该酶能独特地催化蝶啶还原为L-苏式二氢生物蝶呤，纯化倍数为533倍，总产率为3%。天然酶的分子量为55 kDa，十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）显示该酶由两个分子量为26 kDa的亚基组成。该酶在pH8.8和50℃时活性最佳。蝶啶和NADPH的表观米氏常数（Km）分别为21和6.2 μM，催化常数（kcat）值为5.0 s-1。双乙酰也可作为底物，Km为4.0 mM。N-乙酰5-羟色胺、N-乙酰多巴胺和褪黑素的抑制作用非常弱。N-乙酰多巴胺的抑制常数（Ki）值测定为400 μM。其N端氨基酸序列为Met-Lys-His-Ile-Leu-Leu-Ile-Thr-Gly-Ala-Xaa-Lys - Lys - Ile - Xaa - Arg - Ala - Ile - Ala - Leu - Glu - Xaa - Ala - Arg - Xaa-Xaa-Xaa-His-His-His-，与其他蝶啶还原酶具有较高的序列相似性。
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