To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214 -227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214 -227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH 2 -terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K m for cholesterol. The k cat of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.The conversion of cholesterol into 7␣-hydroxycholesterol represents the first and rate-limiting step in overall bile acid biosynthesis, the major pathway for cholesterol elimination in mammals. This reaction takes place in the liver and is catalyzed by the microsomal enzyme cytochrome P450 7A1 (P450 7A1) (1, 2). Human P450 7A1 has been cloned (3) and expressed in Escherichia coli as a truncated (⌬2-24) protein (4); however, little is known about the molecular basis for substrate specificity in this important P450.Based on available crystal structures of eight different P450s that belong to distinct gene families (5-12), a paradigm is beginning to emerge: the mechanism for substrate specificity in the P450 superfamily is complex and determined not solely by residues located inside the enzyme active site (13-15). Unlike many enzymes, the substrate binding pocket of structurally characterized P450s does not form an open cleft at the molecular surface, but rather is buried inside the molecule. A channel connecting the protein exterior and the substrate binding site (substrate access channel) is seen in the structure of only two P450s, 102 and 51 (6, 12), and it is postulated that substrate entry in P450s is controlled by the opening motion of the substrate access channel (16). Although it is not clear how hydrophobic substrates reach the active site of soluble P450s, it has been proposed that in membrane-bound P450s the entrance of the substrate access channel is located wi...

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The cytochrome P450 superfamily of enzymes catalyzes a broad range of oxidative processes involved in the metabolism of fatty acids, biosynthesis of sterols, and elimination of drugs and xenobiotics. Application of the unique properties of P450 enzymes as fine biocatalysts in biotechnology is limited due to their thermal instability and the requirement for auxiliary electron-donor proteins and cofactors. CYP119, a thermophilic P450 enzyme from Sulfolobus solfataricus, was characterized some time ago, but no high-temperature redox partners have been available for it. Here we report reconstitution of CYP119 with a novel high-temperature electron-donor system consisting of a ferredoxin and 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii strain 7 that, unlike all other known P450 electron-donor partners, utilizes coenzyme-A and pyruvic acid rather than NADH or NADPH as the source of electrons. The oxidation of lauric acid by the reconstituted system increased 16-fold as the temperature increased from 25 to 70 degrees C and was functional for more than 30 min at the higher temperature. This first in vitro high-temperature P450 catalytic system is a key step in the development of practical high-temperature monooxygenase systems.

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membranebound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the puta-