No abstract

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G 25 S, D 335 X (W, F, P), and G 210 A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G 30 S and L 37 P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA 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 R 269 G 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.

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.Squalene epoxidase (SE) is an essential flavin adenine dinucleotide (FAD)-dependent monooxygenase in sterol biosynthesis. The fungal enzyme is the target for the allylamines terbinafine and naftifine which inhibit SE in a noncompetitive manner (11). The homology model of SE (Erg1p) of Saccharomyces cerevisiae (9) shows a two-domain structure typical for this class of flavin-dependent enzymes (12): the FAD binding domain (Fig. 1A, lower part, pink) with the characteristic nucleotide binding ␣␤-fold (1, 9, 13), and a second domain typically referred to asthe substrate binding domain (Fig. 1A, FIG. 1. Modeled structure of Erg1p from S. cerevisiae based on the crystal structure of p-hydroxybenzoate hydroxylase. (A) Schematic representation of the three-dimensional model. The cofactor binding domain is shown in pink, the substrate binding domain in blue. The FAD cofactor is depicted as a yellow stick model. The two conserved regions are highlighted in green (motif-1) and cyan (motif-2). (B) Close-up view of motif-1. Amino acid side chains for which mutations are described in the text are displayed as CPK models (C, orange; N, blue; O, red). (C) Close-up view of motif-2. This figure was prepared using PyMol software