Abstract
O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed.
Highlights
Sulfur is a fundamental component of many biomolecules, from amino acids to cofactors and compounds that control the redox homoeostasis
On the basis of the spectral and kinetic data obtained by monitoring the reactivity of triF-Ala with O-acetylserine sulfhydrylase (OASS) and taking into account reaction schemes previously proposed in the reaction of triF-Ala with other pyridoxal 50-phosphate (PLP)-dependent enzymes[20,22,24,25,29,30,36], we propose an inactivation mechanism for OASS-A that is similar to the mechanism proposed for alanine racemase[30] (Scheme 2, path B)
The search for reversible and irreversible inhibitors of OASS-A and OASS-B is dictated by the relevance of these enzymes in the biosynthesis of cysteine in bacteria and by their absence in mammals
Summary
Sulfur is a fundamental component of many biomolecules, from amino acids to cofactors and compounds that control the redox homoeostasis. OASS is present in bacteria as two isoforms, OASS-A and OASSB, named CysK and CysM after the coding genes. Because SAT binding to OASS-A involves anchoring of its C-terminus in OASS-A active site, complex formation leads to the competitive inhibition of OASS-A activity. Both isoforms are present in bacteria but not in humans. Bacteria knocked-out for cysK and cysM exhibit a phenotype with reduced virulence, compromised fitness, and decreased antibiotic resistance[9,10]. For these reasons, OASS has been the target of multiple medicinal chemistry efforts to identify reversible inhibitors potentially useful as antibiotics or enhancers of antibiotic activity[11,12].
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