Role of homoserine transacetylase as a new target for antifungal agents.
Microbial amino acid biosynthesis is a proven yet underexploited target of antibiotics. The biosynthesis of methionine in particular has been shown to be susceptible to small-molecule inhibition in fungi. The first committed step in Met biosynthesis is the acylation of homoserine (Hse) by the enzyme homoserine transacetylase (HTA). We have identified the MET2 gene of Cryptococcus neoformans H99 that encodes HTA (CnHTA) by complementation of an Escherichia coli metA mutant that lacks the gene encoding homoserine transsuccinylase (HTS). We cloned, expressed, and purified CnHTA and determined its steady-state kinetic parameters for the acetylation of L-Hse by acetyl coenzyme A. We next constructed a MET2 mutant in C. neoformans H99 and tested its growth behavior in Met-deficient media, confirming the expected Met auxotrophy. Furthermore, we used this mutant in a mouse inhalation model of infection and determined that MET2 is required for virulence. This makes fungal HTA a viable target for new antibiotic discovery. We screened a 1,000-compound library of small molecules for HTA inhibitors and report the identification of the first inhibitor of fungal HTA. This work validates HTA as an attractive drug-susceptible target for new antifungal agent design.
Nazi
Ishac
I
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Scott
Adam
A
Sham
Anita
A
Rossi
Laura
L
Williamson
Peter R
PR
Kronstad
James W
JW
Wright
Gerard D
GD
D000123:Acetyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D051379:Mice; D008969:Molecular Sequence Data
D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D000123:Acetyltransferases; C021369:homoserine O-acetyltransferase