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Role of Arg458 of mouse CYP27B1 was studied in the interaction of mouse mitochondrial CYP27B1 with adrenodoxin (ADX). It was found that Arg458 of mouse CYP27B1 is conserved in all mitochondrial P450s indicating that this conserved Arg plays an important role in the structural changes of the substrate-binding site of the oxyferic form of mitochondrial P450 to promote monooxygenation of the substrate.

An examination of the rotational diffusion of the fusion proteins of rat liver CYP1A1 and NADPH-cytochrome P450 that were genetically expressed in yeast microsomal membranes was conducted via flash-induced absorption anisotrophy. The large mobile population of the fusion protein, observed to be closer to that of CYP1A1 coexpressed with P450 reductase and more than in CYP1A1 alone, provides proof that CYP1A1 is mobilized by the formation of P450 reductase associations in microsomal membranes.

Bovine [P450.sub.17[Alpha],lyase] containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone ([[Delta].sup.5]-steroid) to 17[Delta]-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17[Alpha]-hydroxylation of progesterone ([[Delta].sup.4]-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17[Alpha]-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17[Alpha]hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17[Alpha]-hydroxyprogesterone was 10 times faster than that of 17[Alpha]-hydroxypregnenolone. The release of the intermediate metabolite of [[Delta].sup.4]-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of [P450.sub.17[Alpha],lyase].

Kinetic studies were conducted on zoxazolamine 6-hydroxylation at 10 degrees centigrade, using the fused enzyme as a catalyst. The fused enzyme between yeast NADPH-P450 reductase and rat P4501Al was constructed. This fused enzyme was introduced into Saccharomyces cerevisiae AH22 cells. Kinetic and spectral studies suggest that the rate limiting step of the reaction might be the release of the product.

A series of single and double mutants are prepared by site-directed mutagenesis focusing on the Arg73 and Arg84 residues of CYP105A1 from Streptomyces griseolus in order to obtain the hyperactive vitamin [D.sub.3] hydroxylase. The structural analyses of single and double mutants have shown that the amino acid residues at positions 73 and 84 have affected the location and conformation of the bound compound in the reaction site and those in the transient binding site.

The crystal structure of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1[alpha], 25-dihydroxyvitamin [D.sub.3] is reported.

The novel metabolites of 25(OH)D3 and 1alpha,25(OH)2D3 by human CYP24A1 are presented. These metabolites appear to be closely related to the C-23 hydroxylation pathway.

Vitamin D3 (VD3), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD3 requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD3 into 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1α,25(OH)2D3. The compound 1α,25(OH)2D3 is positioned 11 Å from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1 -VD3 complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B′ and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1α-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD3. [ABSTRACT FROM AUTHOR]

Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-6165]. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1. We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3). Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3).