PMID- 10518789
OWN - NLM
STAT- MEDLINE
DCOM- 19991214
LR  - 20190620
IS  - 0014-2956 (Print)
IS  - 0014-2956 (Linking)
VI  - 265
IP  - 3
DP  - 1999 Nov
TI  - Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase
      coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia
      coli.
PG  - 950-6
AB  - 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.
FAU - Sawada, N
AU  - Sawada N
AD  - Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
      University, Japan.
FAU - Sakaki, T
AU  - Sakaki T
FAU - Kitanaka, S
AU  - Kitanaka S
FAU - Takeyama, K
AU  - Takeyama K
FAU - Kato, S
AU  - Kato S
FAU - Inouye, K
AU  - Inouye K
LA  - eng
PT  - Journal Article
PL  - England
TA  - Eur J Biochem
JT  - European journal of biochemistry
JID - 0107600
RN  - 0 (DNA, Complementary)
RN  - 0 (Recombinant Proteins)
RN  - 12687-22-8 (Adrenodoxin)
RN  - 1C6V77QF41 (Cholecalciferol)
RN  - EC 1.14.13.13 (25-Hydroxyvitamin D3 1-alpha-Hydroxylase)
RN  - EC 1.18.1.2 (Ferredoxin-NADP Reductase)
SB  - IM
MH  - 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/chemistry/*genetics/*metabolism
MH  - Adrenodoxin/genetics
MH  - Animals
MH  - Cholecalciferol/metabolism
MH  - Cloning, Molecular
MH  - DNA, Complementary/genetics
MH  - Escherichia coli/genetics
MH  - Ferredoxin-NADP Reductase/genetics
MH  - Gene Expression
MH  - Humans
MH  - In Vitro Techniques
MH  - Kinetics
MH  - Mice
MH  - Mutagenesis, Site-Directed
MH  - Recombinant Proteins/chemistry/genetics/metabolism
MH  - Rickets/genetics/metabolism
MH  - Substrate Specificity
EDAT- 1999/10/16 00:00
MHDA- 1999/10/16 00:01
CRDT- 1999/10/16 00:00
PHST- 1999/10/16 00:00 [pubmed]
PHST- 1999/10/16 00:01 [medline]
PHST- 1999/10/16 00:00 [entrez]
AID - ejb794 [pii]
AID - 10.1046/j.1432-1327.1999.00794.x [doi]
PST - ppublish
SO  - Eur J Biochem. 1999 Nov;265(3):950-6. doi: 10.1046/j.1432-1327.1999.00794.x.