ABSTRACT Saccharomyces cerevisiae squalene epoxidase contains two highly conserved motifs, 1 and 2, of unknown function. Amino acid substitutions in both regions reduce enzyme activity and/or alter allylamine sensitivity. In the homology model, these motifs flank the flavin adenine dinucleotide cofactor and form part of the interface between cofactor and substrate binding domains.

ABSTRACTSqualene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies top-hydroxybenzoate hydroxylase (PHBH) fromPseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of theERG1gene, which codes for theSaccharomyces cerevisiaeSE, we isolatederg1alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed inerg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase inerg1mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.