Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: insights into substrate specificity and membrane bound structure-function.

Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A', A, and F-helices, beta3a strand, and beta5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membrane-associated domains.

Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies.

Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates in different pathways of cholesterol degradation as well as in the activation of vitamin D(3). Several approaches were utilized to investigate how two physiological substrates, cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing binding of the two substrates and five substrate analogues followed by computer modeling and site-directed mutagenesis.

Cholesterol binding to cytochrome P450 7A1, a key enzyme in bile acid biosynthesis.

The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7A1) initiates the major pathway for cholesterol elimination in mammals. In the present work we focused on identification of determinants of the CYP7A1 substrate specificity inside the active site using a homology model with a novel P450-fold, site-directed mutagenesis, and substrate-binding and kinetic studies. Forty-one mutants, encompassing twenty-six amino acid residues, were generated and characterized, and of these, seven residues appear to determine cholesterol binding in the active site.

Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9).

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity.

Sequence alignments, variabilities, and vagaries.

It seems as if the algorithms and weighting matrices for multiple sequence alignments of the highly divergent members of the P450 gene superfamily have advanced to the point that unknown proteins can be aligned to structurally known members with reasonable accuracy. As stated earlier, the alignment tends to break down at gaps in the sequence alignments, but these regions can be improved manually. This type of alignment and analysis is especially useful for extracting and analyzing the various genome databases.

Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin: evaluating site-directed mutations by molecular modeling.

The present study was undertaken to evaluate the role of positively charged amino acid residues proposed to reside on the proximal surface of bovine cytochrome P450 cholesterol side chain cleavage (P450scc, CYP11A1) and to determine which residues may be involved in protein-protein interactions with the electron carrier adrenodoxin (Adx). In previous studies, nine different lysine residues were identified by chemical and immunological cross-linking experiments as potentially interacting with Adx, while in the present study, two arginine residues have been identified from sequence alignments. From these 11 residues, 13 different P450scc mutants were made of which only seven were able to be expressed and characterized.