Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 mumol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D-2 and vitamin D-3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D-3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1 The major metabolite of vitamin D-2 by CYP105A1 was 25(OH)D-2, while the major metabolites by CYP27A1 were both 24(OH)D-2 and 27(OH)D-2. These results suggest that CYP105A1 recognizes both vitamin D-2 and vitamin D-3 in a similar manner, while CYP27A1 does not. The K-m values of CYP105A1 for vitamin D-2 25-hydroxylation, vitamin D-3 25-hydroxylation, and 25-hydroxyvitamin D-3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 muM, respectively, suggesting a high affinity of CYP105A1 for these substrates. (C) 2004 Elsevier Inc. All rights reserved.