Molecular cloning of a Schizosaccharomyces pombe cDNA encoding lanosterol synthase and investigation of conserved tryptophan residues.
A Schizosaccharomyces pombe cDNA encoding lanosterol synthase was cloned by complementing a Saccharomyces cerevisiae lanosterol synthase mutant. The predicted 83-kDa protein is 54-58% identical to other lanosterol synthases. The previously known lanosterol synthases contain 229 conserved residues, which should encompass the catalytically essential amino acids. This number is decreased dramatically by including the Sc. pombe lanosterol synthase in the analysis; 42 residues are no longer conserved and therefore are catalytically nonessential. We have begun mutagenic studies to identify catalytic residues from the remaining conserved residues. Mutant Sa. cerevisiae lanosterol synthase genes were generated in which phenylalanine was specifically substituted for conserved tryptophan residues. All of the resultant mutant enzymes retained the ability to complement the Sc. cerevisiae lanosterol synthase mutant, suggesting that these conserved tryptophan residues are not catalytically essential.
Corey
E J
EJ
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA.
Matsuda
S P
SP
Baker
C H
CH
Ting
A Y
AY
Cheng
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D015723:Gene Library; D006801:Humans; D019751:Intramolecular Transferases; D007535:Isomerases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014364:Tryptophan; D007535:Isomerases; D019751:Intramolecular Transferases; C021924:lanosterol synthase