Cloning and expression of squalene epoxidase from the pathogenic yeast Candida albicans.
The allylamine antimycotic terbinafine prevents the formation of sterols by specifically inhibiting squalene epoxidase (SE). The biological and biochemical action of terbinafine on fungal pathogens has been well investigated, but little is known at the molecular level. Here we report the cloning, sequencing and expression of the target of terbinafine from the major pathogen Candida albicans. A C. albicans genomic DNA library was constructed in gamma ZAP Express and screened with a DNA fragment obtained by polymerase chain reaction with two primers designed from sequences common to Saccharomyces cerevisiae and rodent SEs. Two types of clone, approximately 3.9 kbp and 4.1 kbp, were isolated. Both contained an identical open reading frame of 1488 nucleotides, while a few sequence differences were found in the flanking regions, suggesting an allelic heterogeneity. The deduced protein sequence of C. albicans SE, 496 amino acids (55324 Da), is 54% and 41% identical to those of S. cerevisiae and rat, respectively. A 1.8-kb transcript was observed on Northern blots of C. albicans mRNA. Polyclonal antibodies, raised against an internal peptide of C. albicans SE, recognized a protein associated with the particulate fraction of M(r) 55000 on Western blots of C. albicans extracts. C. albicans SE was overexpressed in S. cerevisiae with the expression vector pYES2. In homogenates from S. cerevisiae overexpressing the C. albicans protein SE activity was 10-fold higher than the endogenous activity from controls.
Favre
B
B
Sandoz Research Institute, Department of General Dermatology, Vienna, Austria.
Ryder
N S
NS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000737:chemistry; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D050603:Squalene Monooxygenase; D014158:Transcription, Genetic
D005656:Fungal Proteins; D010105:Oxygenases; D050603:Squalene Monooxygenase