Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme.
Squalene synthase is the first pathway-specific enzyme in the cholesterol biosynthetic pathway. The gene (ERG9) encoding squalene synthase in Saccharomyces cerevisiae has been isolated and characterized (S. M. Jennings, Y. H. Tsay, T. M. Fisch, and G. W. Robinson, 1991, Proc. Natl. Acad. Sci. USA 88, 6038-6042: M. Fegueur, L. Richard, A. D. Charles, F. Karst, 1991, Curr. Genet. 20, 365-372). The structural gene for the enzyme was modified by the polymerase chain reaction to remove a hydrophobic C-terminal domain, and the open reading frame for the truncated protein was cloned into yeast and Escherichia coli expression vectors. In E. coli, overexpressed truncated squalene synthase was soluble and constituted 2-5% of total cellular protein. The recombinant enzyme was purified to > 95% homogeneity in two steps by chromatography on hydroxyapatite and phenyl-Superose. Soluble truncated squalene synthase is monomeric and catalyzes the two-step conversion of farnesyl diphosphate (FPP) to squalene via presqualene diphosphate in the presence of Mg2+ and NADPH. The concentration of FPP needed for half-maximal activity was 40 microM. At higher concentrations, FPP was an inhibitor. The activity of squalene synthase was stimulated by detergents and reached a maximal value of kcat = 3.3 s-1 at 100 microM FPP in the presence of 1% (v/v) Tween 80.
Zhang
D
D
Department of Chemistry, University of Utah, Salt Lake City 84112.
Jennings
S M
SM
Robinson
G W
GW
Poulter
C D
CD
D001483:Base Sequence; D003001:Cloning, Molecular; D005033:Ethylmaleimide / Q000494:pharmacology; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017384:Sequence Deletion; D012995:Solubility; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013186:Farnesyl-Diphosphate Farnesyltransferase; D008274:Magnesium; D005033:Ethylmaleimide