MOLECULAR-CLONING OF A SCHIZOSACCHAROMYCES-POMBE CDNA-ENCODING LANOSTEROL SYNTHASE AND INVESTIGATION OF CONSERVED TRYPTOPHAN RESIDUES

A Schizosacchnromyces pombe cDNA encoding lanosterol synthase was clon ed by complementing a Saccharomyces cerevisiae lanosterol synthase mut ant. The predicted 83-kDa protein is 54-58% identical to other lanoste rol synthases. The previously known lanosterol synthases contain 229 c onserved 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 c onserved residues. Mutant Sa. cerevisiae lanosterol synthase genes wer e generated in which phenylalanine was specifically substituted for co nserved tryptophan residues. All of the resultant mutant enzymes retai ned the ability to complement the Sc. cerevisiae lanosterol synthase m utant, suggesting that these conserved tryptophan residues are not cat alytically essential. (C) 1996 Academic Press, Inc.