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 pH 8.8 and 50 °C. Apparent Km values for sepiapterin and NADPH were 21 and 6.2 μM, 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 μM. 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.

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