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J. Biol. Chem., Vol. 280, Issue 18, 17786-17791, May 6, 2005

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Functional Analysis of Active Site Residues of the Fosfomycin Resistance Enzyme FosA from Pseudomonas aeruginosa^*

Zanna Beharry and Timothy Palzkill

From the Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas 77030

The metalloglutathione transferase FosA catalyzes the conjugation of glutathione to carbon-1 of the antibiotic fosfomycin, rendering it ineffective as an antibacterial drug. Codon randomization and selection for the ability of resulting clones to confer fosfomycin resistance to Escherichia coli were used to identify residues critical for FosA function. Of the 24 codons chosen for randomization, 16 were found to be essential because only the wild type amino acid was selected. These included ligands to the Mn²⁺ and the K⁺, residues that furnish hydrogen bonds to fosfomycin, and residues located in a putative glutathione/fosfomycin-binding site. The remaining eight positions randomized were tolerant to substitutions. Site-directed mutagenesis of some of the essential and tolerant amino acids to alanine was performed, and the activity of the purified proteins was determined. Mutation of the residues that are within hydrogen bonding distance to the oxirane or phosphonate oxygens of fosfomycin resulted in variants with very low or no activity. Mutation of Ser⁹⁴, which bridges one of the phosphonate oxygens with a potassium ion, resulted in insoluble protein. The Y39A mutation in the putative glutathione-binding site resulted in a 4-fold increase in the apparent K_m for glutathione. Only two of the amino acids in the substrate-binding site are conserved in the related fosfomycin resistance proteins FosB and FosX, whereas no amino acids in the putative glutathione-binding site are conserved.

Received for publication, January 28, 2005 , and in revised form, February 28, 2005.

^* This work was supported by National Institutes of Health Grant AI32956 (to T. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-5609; Fax: 713-798-7375; E-mail: timothyp{at}bcm.tmc.edu.

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