title: Site-directed mutagenesis of conserved aromatic residues in rat squalene epoxidase.
abstract: Squalene epoxidase catalyzes the conversion of squalene to (3S)2,3-oxidosqualene, which is a rate-limiting step of the cholesterol biogenesis. To evaluate the importance of conserved aromatic residues, 15 alanine-substituted mutants were constructed and tested for the enzyme activity. Except F203A, all the mutants significantly lost the enzyme activity, confirming the importance of the residues, either for correct folding of the protein, or for the catalytic machinery of the enzyme. Further, interestingly, F223A mutant no longer accepted (3S)2,3-oxidosqualene as a substrate, while Y473A mutant converted (3S)2,3-oxidosqualene to (3S,22S)2,3:22,23-dioxidosqualene twice more efficiently than wild-type enzyme. It is remarkable that the single amino acid replacement yielded mutants with altered substrate and product specificities. These aromatic residues are likely to be located at the substrate-binding domain of the active-site, and control the stereochemical course of the enzyme reaction.
authors.affiliation: School of Pharmaceutical Sciences, The 21st Century COE Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. abei@ys7.u-shizuoka-ken.ac.jp
authors.forename: Ikuro
authors.initials: I
authors.lastname: Abe
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Abe
authors.forename: Weiwei
authors.initials: W
authors.lastname: Lou
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Masuoka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Noguchi
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D050603:Squalene Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D050603:Squalene Monooxygenase; D000409:Alanine