CYP27Al catalyzes vitamin D-3 25-hydroxylation and further hydroxylation at C-1 alpha, C-24 or C-26(27). Molecular modeling of human CYP27Al and docking with 25-hydroxyvitamin D-3 predicted that Gln 85 might be important for 1 alpha-hydroxylation activity of CYP27Al by forming a hydrogen bond with the 25-OH group of 25-hydroxyvitamin D-3. Expectedly, the mutant Q85H expressed in Escherichia coli showed no detectable I ot-hydroxylation activity toward 25-hydroxyvitamin D-3. In addition, Q85H prefers 24-hydroxylation toward 25-hydroxyvitamin D3 whereas the wild-type prefers 26(27)-hydroxylation. A molecular modeling study also suggests that Gln 85 of CYP27Al simultaneously interacts with Asn 107 and the hydroxyl group of the substrate. The fact that Q85L did not contain a heme molecule suggests that the hydrogen bond between Gln 85 and Asn 107 is important for protein folding of CYP27Al. Based on these results, it is possible that Gln 85 plays essential roles in both substrate-binding and protein folding. (c) 2007 Elsevier Inc. All rights reserved.