Abstract
The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.
Highlights
In bacteria and plants cysteine is the only source of sulfur that is required for the synthesis of a variety of biomolecules, including methionine, Fe-S clusters, thiamine, glutathione, and biotin [1,2]
Inactivation of enzymes involved in cysteine and methionine biosynthesis in Mycobacterium tuberculosis, significantly reduces bacterial virulence and persistence during the chronic phase of infection in mice [22]
Most of the residues of the first active site shell are conserved, with residues belonging to the N-terminal domain showing a 90% identity, residue P67 being substituted by A69 in O-acetylserine sulfhydrylases (OASS)-B
Summary
In bacteria and plants cysteine is the only source of sulfur that is required for the synthesis of a variety of biomolecules, including methionine, Fe-S clusters, thiamine, glutathione, and biotin [1,2]. The cysteine regulon of pathogenic microorganisms is up-regulated, in vitro, under oxidative stress [6], in the presence of nitric oxide [7], and in vivo, during infection or long term survival [8,9] It has been proposed and experimentally proved that enzymes involved in sulfur metabolism, and in cysteine biosynthesis, are targets for the development of novel antibiotics [4,6,10,11,12,13,14,15,16,17,18,19,20,21,22,23]. Therapeutic strategies against microbes that rely heavily on sulphur metabolism for efficient host infection and colonization, such as M. tuberculosis and Entamoeba histolytica, have been proposed [4,11]
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