Distinct and potent vitamin D hydroxylation activity acquired by the CYP3A4 I301T single amino acid substitution...

The cytochrome P450 3A4 (CYP3A4) variant I301T has been associated with type 3 rickets, which is characterized by reduced serum calcium and significantly decreased levels of 25-hydroxyvitamin D3 [25(OH)D3] and 1α,25-dihydroxyvitamin D3 [1α,25(OH)₂D3]. Although enhanced 4-hydroxylation of these metabolites was previously proposed as the underlying mechanism causing the disease, the precise enzymatic basis remained unclear. In this study, we investigated the enzymatic properties of CYP3A4-I301T using membrane fractions from recombinant Escherichia coli expressing the variant. Surprisingly, we found that CYP3A4-I301T efficiently produced additional metabolites compared to the wild-type CYP3A4, including 11α,25-dihydroxyvitamin D3 and 1α,11α,25-trihydroxyvitamin D3, which have much lower affinity for the vitamin D receptor than do 25(OH)D3 and 1α,25(OH)₂D3, respectively. Docking simulations suggested that the 3β-hydroxyl group of 25(OH)D3 forms a hydrogen bond with Thr301 of CYP3A4-I301T, while the 25-hydroxyl group interacts with Arg372 and Glu374, favoring hydroxylation at the 11α-position. These results indicate that the I301T mutation confers a gain-of-function phenotype on CYP3A4, enhancing its ability to metabolize 25(OH)D3 and 1α,25(OH)₂D3 into low-activity derivatives. This enzymatic shift likely contributes to substrate depletion and impaired calcium homeostasis in affected individuals. Our findings provide new mechanistic insight into the pathological consequences of a single amino acid substitution in CYP3A4 and expand the understanding of enzyme gain-of-function mutations in metabolic disorders.