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Antimicrobial Agents and Chemotherapy, January 2007, p. 275-284, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00988-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants{triangledown}

Christoph Ruckenstuhl,1 Silvia Lang,1 Andrea Poschenel,1 Armin Eidenberger,1 Pravas Kumar Baral,2 Peter Kohút,3 Ivan Hapala,3 Karl Gruber,2 and Friederike Turnowsky1*

Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Graz, Austria,1 Institute of Chemistry, Karl-Franzens-Universität Graz, Graz, Austria,2 Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic3

Received 8 August 2006/ Returned for modification 11 September 2006/ Accepted 5 October 2006

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 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 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 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.

* Corresponding author. Mailing address: Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria. Phone: 43 316 380 5623. Fax: 43 316 380 9898. E-mail: friederike.turnowsky{at}uni-graz.at.

{triangledown} Published ahead of print on 16 October 2006.

Antimicrobial Agents and Chemotherapy, January 2007, p. 275-284, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00988-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ruckenstuhl, C., Poschenel, A., Possert, R., Baral, P. K., Gruber, K., Turnowsky, F. (2008). Structure-Function Correlations of Two Highly Conserved Motifs in Saccharomyces cerevisiae Squalene Epoxidase. Antimicrob. Agents Chemother. 52: 1496-1499 [Abstract] [Full Text]  


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