Alpha-hydroxylation Of 25-hydroxyvitamin D3 Research Articles

Enzymatic studies on the key enzymes of vitamin D metabolism; 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24)

The key enzymes of vitamin D3 metabolism, renal 25-hydroxyvitamin D3 1 alpha-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24) were expressed in Escherichia coli, and their enzymatic properties were revealed. As expected, mouse CYP27B1 and human CYP27B1 showed the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with the Michaelis constant, Km, value of 2.7 microM. Unexpectedly, both mouse CYP27B1 and human CYP27B1 showed greater Vmax/Km values toward 24,25-dihydroxyvitamin D3 than 25-hydroxyvitamin D3, suggesting that 24, 25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for both CYP27B1. Enzymatic studies on substrate specificity of CYP27B1 revealed that 25-hydroxyl group of vitamin D3 was essential for the 1 alpha-hydroxylase activity, and 24-hydroxyl group enhanced the activity, but, 23-hydroxyl group greatly reduced the activity. On rat CYP24, it was demonstrated that CYP24 catalyzed four-step monooxygenation towards 25-hydroxyvitamin D3. Furthermore, in vivo and in vitro metabolic studies on 1 alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyzed six-step monooxygenation to convert 1 alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1 alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is highly responsible for the metabolism of both 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3. In addition, we have succeeded in the construction of mitochondrial P450 electron transport chain consisting of ADR, ADX and each of CYP27B1 and CYP24 in E. coli cells. The coexpression system with CYP27B1 might be useful as a bioreactor to produce 1 alpha,25-dihydroxyvitamin D3. In contrast, the coexpression system with CYP24 would be applied to metabolic studies of vitamin D analogs used as drugs.

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.

Selective modulation by vitamin D of renal response to parathyroid hormone: a study in calcitriol-resistant rickets.

Calcitriol-resistant rickets (CRR) is an autosomal recessive disease caused by mutated nonfunctioning vitamin D receptors. Because of their lack of biological activity of vitamin D, CRR patients were studied to investigate whether vitamin D modulates the effects of PTH on renal tubules. Five patients with CRR and three controls were studied. After normalization of serum calcium, phosphorus, and PTH levels by oral and i.v. administration of calcium, exogenous PTH-(1-34) was infused, and timed fractions of urine were collected for measurements of cAMP, sodium, potassium, phosphorus, calcium, and bicarbonate. Serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was measured before and after PTH-(1-34) infusion. Urinary cAMP and fractional excretion of potassium, phosphorus, and bicarbonate were similar in CRR patients and controls, as was the rise in the serum 1,25-(OH)2D3 concentration after PTH-(1-34) infusion. However, urinary excretion of calcium and sodium decreased after PTH-(1-34) infusion in controls, but not in CRR patients. These results suggest a selective modulation by vitamin D of the renal response to PTH; 1,25-(OH)2D3 facilitates PTH-induced calcium and sodium reabsorption, but does not influence PTH-induced cAMP excretion; phosphorus, potassium, and bicarbonate tubular transport, or 1 alpha-hydroxylation of 25-hydroxyvitamin D3.

Liver mitochondrial cytochrome P450 CYP27 and recombinant-expressed human CYP27 catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3.

A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig liver mitochondria. It also catalyzed 27-hydroxylation of 25-hydroxyvitamin D3 and 25-hydroxylation of vitamin D3. The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification. Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raised against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxylase activities. Apparently homogeneous preparations of CYP27 from pig and rabbit liver mitochondria catalyzed 1 alpha-hydroxylation. Human liver mitochondrial CYP27 was expressed from its cDNA in Escherichia coli. The nucleotide sequence encoding the N terminus of CYP27 was modified in the first eight codons to achieve expression in E. coli. The purified recombinant-expressed CYP27 reconstituted with the electron-transferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylation of 25-hydroxyvitamin D3. Expression of unmodified CYP27 cDNA in simian COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydroxyvitamin D3.

Overexpression in Escherichia coli and affinity purification of chick kidney ferredoxin.

Vertebrate ferredoxins are 12-14-kDa iron-sulfur proteins, some of which transfer electrons to mitochondrial cytochrome P450s. The function of many of these cytochrome P450s is to catalyze stereospecific hydroxylation of endogenous steroids. As part of our interest in the kidney mitochondrial 1 alpha-hydroxylation of 25-hydroxyvitamin D3, we have constructed an expression plasmid coding for a fusion protein containing the chick kidney ferredoxin. We subcloned chick kidney ferredoxin cDNA, obtained from our vitamin D-deficient chick kidney library by polymerase chain reaction (Brandt, M. E., Gabrik, A. H., and Vickery, L. E. (1991) Gene (Amst.) 97, 113-117) into Qiagen's pQE9, which contains an N-terminal 6xHis tag (peptide sequence for 6 adjacent histidines present in the recombinant proteins). The coding sequence was preceded by a factor Xa cleavage site. The resulting plasmid, pQTcFdx, was overexpressed in Escherichia coli, and the soluble fusion protein was purified from the cell lysate in one step by Ni(II)-nitrilotriacetic acid-agarose chromatography. We obtained 7-10 mg of greater than 99% homogeneous fusion protein from a 1-liter culture and 4-6 mg of mature ferredoxin cleaved by factor Xa. The fusion protein possessed an absorption spectrum and an electron paramagnetic resonance spectrum quantitatively indistinguishable from those published for ferredoxin purified from adrenal glands and placenta or expressed in E. coli with another vector. The fusion protein was active in supporting the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in a reconstitution assay of a solubilized, partially purified preparation of cytochrome P450 from vitamin D-deficient chick kidney. We conclude that the procedure described here is an efficient way to produce and purify vertebrate ferredoxin; the [2Fe-2S] cofactor is assembled in vivo and effectively incorporated into the fusion protein in E. coli; slight alterations at the N terminus do not alter incorporation of the [2Fe-2S] cofactor or the biological activity of ferredoxin, and post-translational modifications, such as phosphorylation, are not an absolute requirement for ferredoxin electron transporting activity. The recombinant ferredoxin can be used for physical studies and other structure-function studies.

Transformation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains

To enzymatically synthesize vitamin D derivatives, we screened about 300 Streptomyces sp. strains. Streptomyces sclerotialus FERM BP-1370 and Streptomyces roseoporus FERM BP-1574 were found to have the ability to convert 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, respectively, to 1 alpha, 25-dihydroxyvitamin D3. The average rates of 1 alpha hydroxylation of 25-hydroxyvitamin D3 were 6.9 micrograms liter-1 min-1 with FERM BP-1370 and 7.0 micrograms liter-1 min-1 with FERM BP-1574. The specific cytochrome P-450 inhibitors carbon monoxide, SKF-525-A, and metyrapone inhibited the hydroxylation of 1 alpha- and 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by FERM BP-1370 and FERM BP-1574. The cytochromes P-450 of these strains were detected by reduced CO difference spectra in the whole-cell suspensions. The appearance of cytochrome P-450 suggests that the cytochromes P-450 of FERM BP-1370 and FERM BP-1574 carry out the hydroxylation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3.

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.

Properties of guinea-pig kidney 25-hydroxyvitamin D3 1 alpha-hydroxylase assayed by isotope dilution-mass spectrometry.

1. A highly specific and accurate method based on isotope dilution-mass spectrometry was used for characterization of the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase in untreated guinea pigs with a normal vitamin D status. In previous work, the properties of the enzyme had been determined in rachitic animals only. 2. With intact mitochondria, the reaction required the presence of citric acid-cycle intermediates. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had an inhibitory effect on the isocitrate-supported reaction, indicating that energy-dependent transhydrogenation is of importance. Mitochondrial respiratory-chain inhibitors (cyanide, rotenone, antimycin A) had no effect on the hydroxylation. CO had an inhibitory effect, suggesting participation of a species of cytochrome P-450 in the reaction. A fraction solubilized from mitochondria by cholate became catalytically active in 1 alpha-hydroxylation of 25-hydroxyvitamin D3 after addition of ferredoxin and ferredoxin reductase. The isocitrate-supported reaction catalysed by crude mitochondria had an apparent Km of about 1 microM. 3. An atmosphere containing 50% O2 was found to be necessary for optimal activity. It is thus possible that O2 may be a limiting factor under normal conditions in vivo. 4. The results demonstrate that the mammalian renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is a cytochrome P-450-dependent mixed-function oxidase with properties similar to those previously reported for the same enzyme system in chicken. The present assay and animal system seem to be suitable for further studies on the mechanism of regulation of the mammalian renal 25-hydroxyvitamin D3 1 alpha-hydroxylase under conditions when the vitamin D status is normal.

Measurement of mammalian 25-hydroxyvitamin D3 24R-and 1 alpha-hydroxylase.

An in vitro assay of mammalian 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases in kidney has been developed. It had been suggested that 25-hydroxyvitamin D binding protein present in mammalian blood and tissues inhibits the enzyme activities in cell-free preparations by binding the substrate 25-hydroxyvitamin D3 more strongly than the hydroxylases bind it. This inhibitory effect is overcome by the addition of substantial amounts of unlabeled 25-hydroxyvitamin D3 to saturate the binding sites of this protein. The resulting metabolites produced in vitro by rat kidney homogenates were isolated and firmly identified by ultraviolet absorption spectrometry and mass spectrometry as 1,25-dihydroxyvitamin D3 and (24R)-24,25-dihydroxyvitamin D3. Maximal 1 alpha-hydroxylation of 25-hydroxyvitamin D3 could be demonstrated in kidney homogenates prepared from vitamin D-deficient rats. Thyroparathyroidectomy of these rats resulted in total suppression of the 1 alpha-hydroxylase. Homogenates of kidney from rats given vitamin D showed little or no 1 alpha-hydroxylase and substantial 24R-hydroxylase activity. Thyroparathyroidectomy of these rts markedly increased the 24R-hydroxylase activity.

The ionic control of 1,25-dihydroxyvitamin D3 production in isolated chick renal tubules.

Isolated renal tubules prepared from vitamin D-deficient chicks catalyze the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 (250HD3) in vitro. The effect of calcium and phosphate on the rate of synthesis of the product, 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3), was studied at two levels: the long-term effects of various dietary calcium and phosphate contents on the ability of the tubules to produce 1, 25 (OH)2D3, and the acute effects of different calcium and phosphate concentrations in the incubation medium on the rate of synthesis of 1,25(OH)2D3 by the tubules. Manipulation of dietary calcium and phosphate sufficient to produce marked changes in the concentration of calcium and phosphate in the serum led to altered rates of 1,25(OH)2D3 synthesis by the isolated renal tubules. The renal tubules isolated from chicks raised on a vitamin D-deficient diet containing 0.43% calcium and 0.3% P as inorganic phosphate showed the highest rate of synthesis of 1,25(OH)2D3. Diets containing more or less of either calcium or phosphate produced chicks whose renal tubules had a slower rate of 1,25(OH)2D3 production. The calcium, phosphate, and hydrogen ion content of the incubation medium were manipulated to determine the possible factors concerned with the immediate regulation of 1,25(OH)2D3 production. A calcium concentration of 0.5-1.0 mM was necessary for optimal enzymatic activity. Concentrations of calcium greater than this optimal concentration inhibited 1,25(OH)2D3 production if phosphate was also present, and this inhibition was more pronounced as the phosphate concentration was increased. The stimulation of 1,25(OH)2D3 production by calcium was less at pH 6.7 than at 7.4. Raising the phosphate concentration from 0 to 6 mM in the absence of calcium also stimulated the rate of synthesis of 1,25(OH)2D3. This stimulatory effect was blocked by 4 mM calcium. However, at 1-2 mM calciu, phosphate had a biphasic influence on 1,25(OH)2D3 production; extracellular concentrations of phosphate from 0.6 to 1.2 mM resulted in less 1,25(OH)2D3 production than higher or lower phosphate concentrations. This biphasic effect was seen both at pH 7.4 and 6.8.