Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli.
We have cloned human 25-hydroxyvitamin D3 1alpha-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 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, 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 1alpha-hydroxylase. These results are quite similar to those of mouse 1alpha-hydroxylase. To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1alpha-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 1alpha-hydroxylase activity by replacement of one amino acid residue.
Sawada
N
N
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Sakaki
T
T
Kitanaka
S
S
Takeyama
K
K
Kato
S
S
Inouye
K
K
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000325:Adrenodoxin / Q000235:genetics; D000818:Animals; D002762:Cholecalciferol / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012279:Rickets / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000325:Adrenodoxin; D002762:Cholecalciferol; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D005287:Ferredoxin-NADP Reductase