The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D-3 and 1alpha-hydroxyvitamin D-3 (1alpha(OH)D-3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D-3 to 1alpha,25(OH)D-3. Both 1alpha-hydroxylation activity toward vitamin D-3, and 25-hydroxylation activity toward 1alpha(OH)D-3 were observed. The K-m and V-max values for 25-hydroxylation activity toward 1alpha(OH)D-3 were estimated to be 1.7 muM and 0.51 mol/min/mol P450, respectively, while those for 1alpha-hydroxylation activity toward 25(OH)D-3 were 0.050 muM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1alpha-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D-3 to 1alpha25(OH)D-3, 1alpha-hydroxylation, and 25-hydroxylation of vitamin D-3 appear to be closely linked together. (C) 2004 Elsevier Inc. All rights reserved.