Enzymatic properties of human 25‐hydroxyvitamin D3 1α‐hydroxylase

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Abstract
We have cloned human 25-hydroxyvitamin D3 1α-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1α-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R),25(OH)2D3 were 2.7 and 1.1 µm, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1α-hydroxylase. These results are quite similar to those of mouse 1α-hydroxylase. To establish a highly sensitive in vivo system, 1α-hydroxylase, adrenodoxin and NADPH–adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1α-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH–adrenodoxin reductase through adrenodoxin to 1α-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1α-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1α-hydroxylase activity by replacement of one amino acid residue.