{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,20]],"date-time":"2026-06-20T05:30:31Z","timestamp":1781933431469,"version":"3.54.5"},"reference-count":27,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2007,5,8]],"date-time":"2007-05-08T00:00:00Z","timestamp":1178582400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Chirality"],"published-print":{"date-parts":[[2007,7]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3\u2032\u2010hydroxylation, and searched the roles of amino acid residues, such as Phe\u2010100, Phe\u2010114, Asp\u2010293, Glu\u2010300, and Phe\u2010476 of CYP2C19 in the stereoselective HB 3\u2032\u2010hydroxylation, using a yeast cell expression system and site\u2010directed mutagenesis method. CYP2C19 wild\u2010type exerted substrate enantioselectivity of (<jats:italic>R<\/jats:italic>)\u2010HB \u226b (<jats:italic>S<\/jats:italic>)\u2010HB and metabolite diastereoselectivity of 3\u2032(<jats:italic>R<\/jats:italic>) &lt; 3\u2032(<jats:italic>S<\/jats:italic>) in 3\u2032\u2010hydroxylation of HB enantiomers. The substitution of Asp\u2010293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide\u2010difference spectrum. CYP2C19\u2010E300A and CYP2C19\u2010E300V with alanine and valine, respectively, in place of Glu\u2010300 exerted total HB 3\u2032\u2010hydroxylation activities of 45 and 108%, respectively, that of the wild\u2010type. Interestingly, these two mutants showed substrate enantioselectivity of (<jats:italic>R<\/jats:italic>)\u2010HB &lt; (<jats:italic>S<\/jats:italic>)\u2010HB, which is opposite to that of the wild\u2010type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe\u2010476 by alanine increased total HB 3\u2032\u2010hydroxylation activity to approximately 3\u2010fold that of the wild\u2010type. Particularly, 3\u2032(<jats:italic>S<\/jats:italic>)\u2010OH\u2010(<jats:italic>S<\/jats:italic>)\u2010HB\u2010forming activity elevated to 7\u2010fold that of the wild\u2010type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu\u2010300 and Phe\u2010476 are important in stereoselective oxidation of HB enantiomers by CYP2C19. Chirality, 2007. \u00a9 2007 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/chir.20412","type":"journal-article","created":{"date-parts":[[2007,5,8]],"date-time":"2007-05-08T21:16:45Z","timestamp":1178659005000},"page":"550-558","source":"Crossref","is-referenced-by-count":5,"title":["Stereoselective hexobarbital 3\u2032\u2010hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 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