title: Sequence requirements for cytochrome P-450IIB1 catalytic activity. Alteration of the stereospecificity and regioselectivity of steroid hydroxylation by a simultaneous change of two hydrophobic amino acid residues to phenylalanine.
abstract: The phenobarbital-inducible P-450 forms IIB1 and IIB2 are identical in sequence except for 14 amino acid differences within the carboxyl-terminal half of the molecule. IIB1 has about a 5-10-fold higher turnover number for most monooxygenase substrates examined although the substrate specificities of both enzymes are virtually identical. Both P-450s oxygenate testosterone to yield the 16 alpha-hydroxy, 16 beta-hydroxy, 17-keto, and 16 beta-hydroxy, 17-keto metabolites as major products. A variant IIB2 cDNA, isolated from an uninduced rat liver lambda gt11 library, and when expressed in Hep G2 cells using a vaccinia virus vector, was found to code for a protein that produced the 16 alpha-hydroxy and 17-keto metabolites of testosterone but no 16 beta-hydroxylated products. Although the published sequences of IIB1 and IIB2 are identical within the N-terminal halves of the proteins, sequence analysis of the variant cDNA revealed two amino acid substitutions in this region; Leu58----Phe and I1e114----Phe. When these two amino acid changes were incorporated into IIB1, via construction of a chimeric cDNA, the resultant expressed enzyme did not catalyze the 16 beta-hydroxylation of testosterone or androstenedione. Formation of the 16 alpha-hydroxy and 17-keto metabolites, however, was only slightly reduced compared with the parent IIB1. A IIB1 protein that possessed only the I1e114----Phe replacement catalyzed the production of all four testosterone metabolites with only slightly different product ratios compared with the parent enzyme. The substrate specificity of a IIB1 variant containing only the Leu58----Phe replacement could not be determined, since that protein did not accumulate in cells infected with the corresponding recombinant vaccinia virus. These data suggest that two distinct amino acid residues located within the amino-terminal fourth of IIB1 and IIB2 can affect substrate orientation at the active site.
authors.affiliation: Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
authors.forename: T
authors.initials: T
authors.lastname: Aoyama
authors.forename: K
authors.initials: K
authors.lastname: Korzekwa
authors.forename: K
authors.initials: K
authors.lastname: Nagata
authors.forename: M
authors.initials: M
authors.lastname: Adesnik
authors.forename: A
authors.initials: A
authors.lastname: Reiss
authors.forename: D P
authors.initials: DP
authors.lastname: Lapenson
authors.forename: J
authors.initials: J
authors.lastname: Gillette
authors.forename: H V
authors.initials: HV
authors.lastname: Gelboin
authors.forename: D J
authors.initials: DJ
authors.lastname: Waxman
authors.forename: F J
authors.initials: FJ
authors.lastname: Gonzalez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002678:Chimera; D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors; D007532:Isoleucine; D007930:Leucine; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine; D011061:Poly A / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics*; D013739:Testosterone / Q000302:isolation & purification / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D007532:Isoleucine; D011061:Poly A; D013739:Testosterone; D010649:Phenylalanine; D013250:Steroid Hydroxylases; D013252:Steroid 11-beta-Hydroxylase; D007930:Leucine