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World Health Organization (WHO) reports that one-third of the world's population is infected with a persistent form of Mycobacterium tuberculosis (M.tb), the causative bacterium responsible for causing the dreaded tuberculosis disease. Targeting mycobacterial persisters is important for achieving WHO's End TB target. The de-novo cysteine biosynthetic pathway is a novel target for addressing M.tb persistence. The two-step pathway comprises of serine acetyltransferase/CysE and O-acetyl-serinesulfhydrylase/OASS/CysK. The present study is an attempt to understand the structural features of mycobacterial CysE by investigating the divergence amongst orthologous through phylogenetic analysis. Mapping of mycobacterial CysE sequences on the whole orthologous (COG1045) tree segregated the species into four clusters and several isoforms leading to their descendants identification. Interestingly the analysis revealed that the extended C-terminal a-helix believed unique to M.tb is also present in other organisms such as: Campylobacter ureolyticus, Bacillus cereus, Geminocystis herdmanii and Paenibacillus borealis. Further, the Hidden Markov model search against the whole Uniprot database suggests a plausible role of C-terminal a-helix of CysE in strengthening the substrate and/or co-factor binding. In addition, phylogenetic analysis of CysE sequences from the Mycobacteriaceae family facilitates grouping them under ten well-formed and six monophyletic clades, each based on characteristic features with respect to domain architecture, oligomeric assembly, C-terminal tetra-peptide tail, regulatory and feedback mechanism etc. Employing molecular phylogeny in conjunction with structural analysis has provided detailed insights for mycobacterial CysEs as drug target. [ABSTRACT FROM AUTHOR]

The antimicrobial resistant strains of several pathogens are major culprits of hospital-acquired nosocomial infections. An active and urgent action is necessary against these pathogens for the development of unique therapeutics. The cysteine biosynthetic pathway or genes (that are absent in humans) involved in the production of L-cysteine appear to be an attractive target for developing novel antibiotics. CysE, a Serine Acetyltransferase (SAT), catalyzes the first step of cysteine synthesis and is reported to be essential for the survival of persistence in several microbes including Mycobacterium tuberculosis. Structure determination provides fundamental insight into structure and function of protein and aid in drug design/discovery efforts. This review focuses on the overview of current knowledge of structure function, regulatory mechanism, and potential inhibitors (active site as well as allosteric site) of CysE. Despite having conserved structure, slight modification in CysE structure lead to altered the regulatory mechanism and hence affects the cysteine production. Due to its possible role in virulence and vital metabolism of pathogens makes it a potential target in the quest to develop novel therapeutics to treat multi-drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) isocitrate lyase (ICL) is an established drug target that facilitates Mtb persistence. Unlike other mycobacterial strains, where ICL2 is a single gene product, H37Rv has a split event, resulting in two tandemly coded icls - rv1915 and rv1916. Our recent report on functionality of individual Rv1915 and Rv1916, led to postulate the cooperative role of these proteins in pathogen's survival under nutrient-limiting conditions. This study investigates the possibility of Rv1915 and Rv1916 interacting and forming a complex. Pull down assay, activity assay, mass spectrometry and site directed mutagenesis was employed to investigate and validate Rv1915-Rv1916 complex formation. Rv1915 and Rv1916 form a stable complex in vitro, with enhanced ICL/MICL activities as opposed to individual proteins. Further, activities monitored in the presence of acetyl-CoA show significant increase for Rv1916 and the complex but not of Rv0467 and Rv1915Δ90CT. Both full length and truncated Rv1915Δ90CT can form complex, implying the absence of its C-terminal disordered region in complex formation. Further, in silico analysis and site-directed mutagenesis studies reveal Y64 and Y65 to be crucial residues for Rv1915-Rv1916 complex formation. This study uncovers the association between Rv1915 and Rv1916 and supports the role of acetyl-CoA in escalating the ICL/MICL activities of Rv1916 and Rv1915Δ90CT-Rv1916 complex. Partitioning of ICL2 into Rv1915 and Rv1916 that associates to form a complex in Mtb H37Rv, suggests its importance in signaling and regulation of metabolic pathway particularly in carbon assimilation. • Rv1915 and Rv1916 of Mtb H37Rv associate together to form in vitro protein-protein complex. • Deletion of disordered C-terminal of Rv1915 does not abrogate complex formation with Rv1916. • Site-directed mutagenesis studies show Y64 and Y65 residues of Rv1916 to be crucial for complex formation. • The presence of acetyl-CoA increases the ICL and MICL activities of Rv1916 and the complex. [ABSTRACT FROM AUTHOR]

• Cloning, purification and biochemical characterization of CysE from Klebsiella pneumoniae • 3-D crystal structure of Kpn CysE complexed with L-cysteine at 2.8 Å • In silico identification and in vitro validation of inhibitory potential of quercetin • Identification of an allosteric site at the trimer-trimer interface in the hexameric CysE The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity. [ABSTRACT FROM AUTHOR]