title: The role of the conserved threonine in P450 BM3 oxygen activation: substrate-determined hydroxylation activity of the Thr268Ala mutant.
abstract: The hydroxylation activity of the Thr268Ala mutant of P450(BM3) has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis-cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished.
authors.affiliation: Chemistry, School of Molecular and Microbial Sciences, University of Queensland, St. Lucia Brisbane 4072, Australia.
authors.forename: Max J
authors.initials: MJ
authors.lastname: Cryle
authors.forename: James J
authors.initials: JJ
authors.lastname: De Voss
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D013912:Threonine; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000409:Alanine; D010100:Oxygen