Sandra E. Graham

A close family resemblance: the importance of structure in understanding cytochromes P450.

Cytochromes P450 comprise a very large superfamily of hemeproteins which generally monooxygenate hydrophobic compounds. P450s appear to have a common conserved structural core, yet are variable in regions involved in substrate recognition and binding, and in redox-partner binding. These differences can be identified by an analysis in which structural alignments and homology models are used to compare the various classes and families of P450s.

Practical, enantiospecific syntheses of 14,15-EET and leukotoxin B (vernolic acid)

Abstract Cytochrome P450BM3 and its F87V mutant were exploited for a convenient, laboratory scale (1 mmol) preparation of 14( S ),15( R )-epoxyeicosatrienoic acid [14( S ),15( R )-EET] from arachidonic acid and (+)-leukotoxin B [(+)-12( S ),13( R )-vernolic acid] from linoleic acid, respectively. Their enantiomers were accessed via a four-step chemical inversion.

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. Variations of the conservation analysis can be used to identify charged and uncharged residues that may be important in domain/domain interactions with redox partners or effector molecules (e.g., cytochrome b5). From these alignments and with comparative analysis within families and across P450 families, one can readily obtain an estimation of those residues that might be involved in substrate binding, in redox partner interaction, and in the catalytic mechanism.

Molecular modeling of mammalian cytochromes P450 : Application to study enzyme function

Cytochromes P450 are important heme-containing enzymes that catalyze the oxidation of a vast array of endogenous and exogenous compounds, including drugs and carcinogens. One of the more successful approaches to study P450 function involves molecular modeling. Because none of the mammalian P450s have been crystallized, a number of homology models have been constructed based on the structures of known bacterial P450s. Molecular models, generated using molecular replacement or distance geometry methods, can be used to dock substrates and/or inhibitors in the active site to explain various aspects of enzyme function. The majority of modeling research has dealt with enzyme-substrate interactions in the active site. The analysis of these interactions has helped us to better understand the mechanism of P450 catalysis and provided the structural basis for the regio- and stereospecificity of substrate oxidation as well as susceptibility to inhibition or inactivation. The models have been utilized to identify and/or confirm key residues and to rationally interpret experimental data. The alteration in activity in a mutant P450 can be related to changes in enzyme-substrate/inhibitor interactions, such as the removal or appearance of van der Waals overlaps or changes in compound mobility. Homology models can also help to analyze P450-redox partner interactions and identify critical determinants of protein stability. We can expect further development of molecular modeling methods and their increasing contribution into research on P450 function as an integral part of a combined theoretical-experimental approach.

The Influence of the Amino Acid Substitution I98K on the Catalytic Activity of Baboon Cytochrome P450 Side‐Chain Cleavage (CYP11A1)

Cytochrome P450 side‐chain cleavage (CYP11A1) catalyzes the first and “rate‐limiting” step in steroidogenesis, the conversion of cholesterol to pregnenolone. In an effort to gain further insight into the structure/function relationship of this key enzyme, CYP11A1 was characterized in the Cape baboon (Papio ursinus), a species closely related to humans. Baboon cDNA was isolated from adrenal tissue and direct sequence analysis showed mature baboon and human CYP11A1 share 98% deduced amino acid homology. The cDNA was subsequently amplified and two recombinant constructs, CYP11A1a and CYP11A1b, were cloned. Sequence analyses of the constructs revealed four amino acid substitutions. The constructs were expressed in nonsteroidogenic mammalian COS‐1 cells with 25‐hydroxycholesterol as substrate. Apparent Km values of 1.62 and 4.53 µM were determined for CYP11A1a and CYP11A1b, respectively. Homology modeling revealed that the lower substrate affinity of CYP11B1b could be attributed to an I98K substitution, which lies between the B and C helices, providing further evidence for the importance of this domain in the catalytic activity of CYP11A1.