The comparative reaction efficiencies of currently used nucleophilic and electrophilic probes towards cysteine sulfenic acid have been thoroughly evaluated in two different settings – (i) a small molecule dipeptide based model and, (ii) a recombinant protein model. We further evaluated the stability of corresponding thioether and sulfoxide adducts under reducing conditions which are commonly encountered during proteomic protocols and in cell analysis. Powered by the development of new cyclic and linear C-nucleophiles, the unsurpassed efficiency in the capture of sulfenic acid under competitive conditions is achieved and thus holds great promise as highly potent tools for activity-based sulfenome profiling.
The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress made in the development of inhibitors of sulfur metabolism enzymes.
N 5 -carboxyaminoimidazole ribonucleotide synthetase (N 5 -CAIR synthetase), a key enzyme in microbial de novo purine biosynthesis, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to N 5 -CAIR. To date, this enzyme has only been observed in microorganisms, and thus it represents an ideal target for antimicrobial drug development. Here we report the cloning, crystallization, and three-dimensional structural analysis of Aspergillus clavatus N 5 -CAIR synthetase solved in the presence of either Mg 2 ATP or MgADP and AIR. These structures, determined to 2.1 Å and 2.0 Å, respectively, revealed that AIR binds in a pocket analogous to that observed for other ATP-grasp enzymes involved in purine metabolism. On the basis of these models, a site-directed mutagenesis study was subsequently conducted that focused on five amino acid residues located in the active site region of the enzyme. These investigations demonstrated that Asp 153 and Lys 353 play critical roles in catalysis without affecting substrate binding. All other mutations affected substrate binding and in some instances, catalysis as well. Taken together, the structural and kinetic data presented here suggest a catalytic mechanism whereby Mg 2 ATP and bicarbonate first react to form the unstable intermediate carboxyphosphate. This intermediate subsequently decarboxylates to CO 2 and inorganic phosphate, and the amino group of AIR, through general base assistance by Asp 153, attacks CO 2 to form N 5 -CAIR.Arguably, one of the most important developments in the history of modern medicine has been the discovery of antibiotics. The golden age of antibacterial drug discovery began during the 1940s and by the beginning of the 1970s, most of the major classes of antibiotics currently in clinical use had been discovered (1,2). The rate of antimicrobial drug discovery has since slowed as evidenced by the fact that only two new classes of antibiotics, the lipopeptides and the oxazolidinones, have been brought to the market within the last 40 years (3,4). Unfortunately, as the pharmaceutical industry's interest in antibiotic drug discovery has waned, resistance to existing antibiotics has grown. Recent studies have shown that approximately 50% of all Staphylococcus aureus infections are methicillin resistant and, strikingly, S. aureus resistance to most other existing antibiotics has been detected in clinical settings (1,5,6 The increasing prevalence of antibiotic resistant infections has led to a recent, renewed interest in antibiotic drug development. For example, studies have examined enzymes such as pantothenate kinase (7), tRNA synthetase (8), and DNA ligase (9), or biosynthetic pathways such as those for the production of isoprenoids (10) and aromatic amino acid synthesis (11), as potential antibacterial drug targets. One unexplored pathway for antimicrobial drug design is de novo purine biosynthesis, which is markedly different in microorganisms than in humans (12-16). As revealed in studies conducted in the 1980s, bacteria, yeast and fungi ...
The increasing risk of drug resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis is different in microbes than in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N 5 -carboxyaminoimidazole ribonucleotide (N 5 CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small molecule inhibitors of N 5 CAIR synthetase have been published. To remedy this problem, we have conducted highthroughput screening (HTS) against Escherichia coli N 5 CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 that inhibited the enzyme. The hits identified could be classified into three classes based upon chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis-Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicate that these compounds do not follow normal MichaelisMenten kinetics. Instead, these compounds inhibit N 5 CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is noncompetitive with both substrates. Finally, these compounds display no inhibition of the human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N 5 CAIR synthetase
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