Enzymatic properties of mouse 25-hydroxyvitamin D3 1 alpha-hydroxylase expressed in Escherichia coli.
Renal 25-hydroxyvitamin D3 1 alpha-hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109. As expected, the bacterially-expressed enzyme catalyzes the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with a Michaelis constant, K(m), value of 2.7 microM. Unexpectedly, the enzyme also hydroxylates the 1 alpha-position of 24,25-dihydroxyvitamin D3 with a K(m) of 1.3 microM, and a fourfold higher Vmax/K(m) compared with the 25-hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for 1 alpha-hydroxylase. In addition, the enzyme showed 1 alpha-hydroxylase activity toward 24-oxo-25-hydroxyvitamin D3. However, it showed only slight activity towards 23,25-dihydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3. These results suggest that the 25-hydroxyl group of vitamin D3 is essential for the 1 alpha-hydroxylase activity and the 24-hydroxyl group enhances the activity, but the 23-hydroxyl group greatly reduced the activity. Another remarkable finding is that living recombinant E. coli cells can convert the substrates into the 1 alpha-hydroxylated products, suggesting the presence of a redox partner of 1 alpha-hydroxylase in E. coli cells.
Sakaki
T
T
Division of Applied Life Sciences, Kyoto University, Japan.
Sawada
N
N
Takeyama
K
K
Kato
S
S
Inouye
K
K
D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism*; D000818:Animals; D002112:Calcifediol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006900:Hydroxylation; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D015394:Molecular Structure; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D015650:24,25-Dihydroxyvitamin D 3; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002112:Calcifediol