Putative Helix F Contributes to Regioselectivity of Hydroxylation in Mitochondrial Cytochrome P450 27A1

Abstract

On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F and G helices of mitochondrial P450s 27A1 and 11A. We introduced substitutions at Phe-207, Ile-211, and Phe-215 within putative helix F and at Trp-235 and Tyr-238 within putative helix G in P450 27A1 and compared wild type and mutants with respect to catalytic activity, product pattern, substrate binding, formation of hydrogen peroxide, and interaction with redox partner. Results indicate that the mutated residues are important for delivery of the correctly oriented substrate to the P450 active site. The I211K and F215K mutations, for example, affected the regioselectivity of P450 27A1-dependent hydroxylation reactions and conferred the P450 capacity to cleave the C-C bond of the substrate during the catalytic cycle. Studies of P450 11A1 indicate that Phe-202 has functions similar to those of its counterpart in P450 27A1 (Phe-215). We propose that putative helices F and G form the sides of the substrate-access channel, thus providing the additional mechanism to control regioselectivity of hydroxylation in mitochondrial P450s.

Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1

“…The K226R substitution reproduces one of the mutations that underlies CTX. Trp-235 and Tyr-238 were studied earlier and the properties of the W235A and Y238A mutants are described elsewhere (15). Table II and Fig.…”

“…The present work is the first study showing that the putative F-G loop and the N-terminal part of the putative G helix are also the sites of membrane attachment in mitochondrial P450 27A1. As this region is amphipathic and about the same size in different mitochondrial P450s (15), it is very likely that the putative F-G loop and a part of the helix G are also involved in membrane binding in other mitochondrial P450s. Thus, microsomal and mitochondrial P450s appear to have an overlapping membrane-binding site.…”

“…It is likely that another cholesterol-metabolizing enzyme, mitochondrial P450 27A1, also recruits cholesterol from the lipid bilayer, as neither the enzyme nor its substrate are soluble in the aqueous environment of the cell. Previously, we have studied the role of the putative helices F and G in P450 27A1, regions that flank the F-G loop (15). Our data indicate that the putative helices F and G form the sides of the of the substrate access channel in this P450, and residues lining the interior of the channel control the orientation of the substrate as it enters the enzyme active site (15).…”

“…Previously, we have studied the role of the putative helices F and G in P450 27A1, regions that flank the F-G loop (15). Our data indicate that the putative helices F and G form the sides of the of the substrate access channel in this P450, and residues lining the interior of the channel control the orientation of the substrate as it enters the enzyme active site (15). Here we describe the effect of mutation of amino acid residues located within the putative F-G loop on subcellular distribution and kinetic parameters for cholesterol 27-hydroxylation of P450 27A1, and we compare mitochondrial P450 27A1 with microsomal P450 7A1.…”

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

“…Compounds are initially made up in a high concentration of cyclodextrin (typically 45%) then can be diluted into culture or assay systems without the substrate precipitating. Our initial studies to determine the K m of P450scc for D3 and cholesterol using a final concentration of 4.5% cyclodextrin gave values orders of magnitude higher than other researchers have reported using this method of substrate solubilization (Guryev et al, 2003;Pikuleva et al, 2001). We therefore tested the effects of the cyclodextrin concentration on K m and k cat for these substrates (Table 3).…”