RESUMO
Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.
Assuntos
Colesterol 7-alfa-Hidroxilase/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Colesterol 7-alfa-Hidroxilase/genética , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Ligação de Hidrogênio , Hidroxilação , Cetocolesteróis/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Oxysterols and neurosteroids are important signaling molecules produced by monooxygenases of the cytochrome P450 family that realize their effect through nuclear receptors. CYP7B1 catalyzes the 6- or 7-hydroxylation of both steroids and oxysterols and thus is involved in the metabolism of neurosteroids and bile acid synthesis, respectively. The dual physiological role of CYP7B1 is evidenced from different diseases, liver failure and progressive neuropathy, caused by enzyme malfunction. Here we present biochemical characterization of CYP7B1 at the molecular level to understand substrate specificity and susceptibility to azole drugs. Based on our experiments with purified enzyme, the requirements for CYP7B1 hydroxylation of steroid molecules are as follows: C5 hydrogen in the α-configuration (or double bond at C5), a polar group at C17, a hydroxyl group at C3, and the absence of the hydroxyl group at C20-C24 in the C27-sterol side chain. 21-hydroxy-pregnenolone was identified as a new substrate, and overall low activity toward pregnanes could be related to the increased potency of 7-hydroxy derivatives produced by CYP7B1. Metabolic conversion (deactivation) of oxysterols by CYP7B1 in a reconstituted system proceeds via two sequential hydroxylations. Two mutations that are found in patients with diseases, Gly57Arg and Phe216Ser, result in apo-P450 (devoid of heme) protein formation. Our CYP7B1 homology model provides a rationale for understanding clinical mutations and relatively broad substrate specificity for steroid hydroxylase.