Discovery of uncompetitive inhibitors of SapM that compromise intracellular survival of Mycobacterium tuberculosis

SapM is a secreted virulence factor from Mycobacterium tuberculosis critical for pathogen survival and persistence inside the host. Its full potential as a target for tuberculosis treatment has not yet been exploited because of the lack of potent inhibitors available. By screening over 1500 small molecules, we have identified new potent and selective inhibitors of SapM with an uncompetitive mechanism of inhibition. The best inhibitors share a trihydroxy-benzene moiety essential for activity. Importantly, the inhibitors significantly reduce mycobacterial burden in infected human macrophages at 1 µM, and they are selective with respect to other mycobacterial and human phosphatases. The best inhibitor also reduces intracellular burden of Francisella tularensis, which secretes the virulence factor AcpA, a homologue of SapM, with the same mechanism of catalysis and inhibition. Our findings demonstrate that inhibition of SapM with small molecule inhibitors is efficient in reducing intracellular mycobacterial survival in host macrophages and confirm SapM as a potential therapeutic target. These initial compounds have favourable physico-chemical properties and provide a basis for exploration towards the development of new tuberculosis treatments. The efficacy of a SapM inhibitor in reducing Francisella tularensis intracellular burden suggests the potential for developing broad-spectrum antivirulence agents to treat microbial infections.

Second distinct conformation of the phosphohistidine loop in succinyl-CoA synthetase

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Å between the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and β-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141β and Glu204β via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

Recent advances in small-molecule HIV-1 integrase inhibitors

: HIV-1 integrase catalyzed the insertion of the viral DNA into the genome of human cells in the process of retrotranscription. Integrase is an attractive target for HIV-1 treatment due to the lack of its homologue in human cells and its vital role in HIV-1 replication. Although a major progress about the development of HIV-1 integrase inhibitors has been made, some thorny problems, such as the drug resistance, led to the further study of HIV-1 integrase inhibitors. This review briefly discussed the structure, function and mechanism of catalysis of HIV-1 integrase and made a further conclusion for recent advances in small-molecule inhibitors of HIV-1 integrase.

Application of Dehalogenase Enzymes in Bioremediation of Halogenated Pollutants

The halogenated hydrocarbons have been widely used by human beings. They are xenobiotic and toxic. The microbes having a specific group of hydrolase enzymes, known as dehalogenases, that actually break the carbon-halogen bonds of the halogenated substances and subsequently convert them into their non-toxic forms. In this chapter, the categories of dehalogenase enzymes possessed by microorganisms are narrated. The overall source, mechanism of catalysis, and structural aspects of the haloalkane dehalogenase enzymes have been discussed with special focus to the bioremediation of 1, 2 dichloroethane.

Generalized enzymatic mechanism of catalysis by tetrameric l-asparaginases from mesophilic bacteria

The mechanism of catalysis by the l-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of l-Asn by the type II l-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric l-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (l-Asp and l-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by l-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to l-Asn and l-Gln, we postulate that it is common for all these structurally related enzymes.

Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.

Structure and mechanism of human diacylglycerol acyltransferase 1

SummaryHuman diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage. The lack of 3-dimensional structure has limited our understanding of substrate recognition and mechanism of catalysis, and hampers rational targeting of hDGAT1 for therapeutic purposes. Here we present the structure of hDGAT1 in complex with a substrate oleoyl Coenzyme A at 3.1 Å resolution. hDGAT1 forms a homodimer and each protomer has nine transmembrane helices that carve out a hollow chamber in the lipid bilayer. The chamber encloses highly conserved catalytic residues and has separate entrances for the two substrates fatty acyl Coenzyme A and diacylglycerol. The N-terminus of hDGAT1 makes extensive interactions with the neighboring protomer, and is required for enzymatic activity.

The eukaryotic tRNA-guanine transglycosylase enzyme inserts queuine into tRNA via a sequential bi–bi mechanism

The mechanism of catalysis associated with the human TGT enzyme has been elucidated and differs from that associated with its eubacterial counterpart.