Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis isocitrate lyase

Mycobacterium tuberculosis isocitrate lyases (ICLs) form a covalent adduct with itaconate through their catalytic cysteine residue. These results reveal atomic details of itaconate inhibition and provide insights into the catalytic mechanism of ICLs.

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Structural insights into the catalytic mechanism of a sacrificial sulfur insertase of the N-type ATP pyrophosphatase family, LarE

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704967114 ◽

2017 ◽

Vol 114 (34) ◽

pp. 9074-9079 ◽

Cited By ~ 16

Author(s):

Matthias Fellner ◽

Benoît Desguin ◽

Robert P. Hausinger ◽

Jian Hu

Keyword(s):

Structural Analysis ◽

Crystal Structures ◽

Lactobacillus Plantarum ◽

Sulfur Atom ◽

Catalytic Mechanism ◽

Cysteine Residue ◽

Sulfur Transfer ◽

Ligand Free ◽

Catalytic Cysteine ◽

Structural Insights

The lar operon in Lactobacillus plantarum encodes five Lar proteins (LarA/B/C/D/E) that collaboratively synthesize and incorporate a niacin-derived Ni-containing cofactor into LarA, an Ni-dependent lactate racemase. Previous studies have established that two molecules of LarE catalyze successive thiolation reactions by donating the sulfur atom of their exclusive cysteine residues to the substrate. However, the catalytic mechanism of this very unusual sulfur-sacrificing reaction remains elusive. In this work, we present the crystal structures of LarE in ligand-free and several ligand-bound forms, demonstrating that LarE is a member of the N-type ATP pyrophosphatase (PPase) family with a conserved N-terminal ATP PPase domain and a unique C-terminal domain harboring the putative catalytic site. Structural analysis, combined with structure-guided mutagenesis, leads us to propose a catalytic mechanism that establishes LarE as a paradigm for sulfur transfer through sacrificing its catalytic cysteine residue.

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Demystifying the catalytic pathway of Mycobacterium tuberculosis isocitrate lyase

Scientific Reports ◽

10.1038/s41598-020-75799-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Collins U. Ibeji ◽

Nor Amirah Mohd Salleh ◽

Jia Siang Sum ◽

Angela Chiew Wen Ch’ng ◽

Theam Soon Lim ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Electrostatic Interactions ◽

Catalytic Mechanism ◽

Isocitrate Lyase ◽

Hydroxyl Group ◽

The Core ◽

Dormant Stage ◽

Lower Activation ◽

Catalytic Cleavage

Abstract Pulmonary tuberculosis, caused by Mycobacterium tuberculosis, is one of the most persistent diseases leading to death in humans. As one of the key targets during the latent/dormant stage of M. tuberculosis, isocitrate lyase (ICL) has been a subject of interest for new tuberculosis therapeutics. In this work, the cleavage of the isocitrate by M. tuberculosis ICL was studied using quantum mechanics/molecular mechanics method at M06-2X/6-31+G(d,p): AMBER level of theory. The electronic embedding approach was applied to provide a better depiction of electrostatic interactions between MM and QM regions. Two possible pathways (pathway I that involves Asp108 and pathway II that involves Glu182) that could lead to the metabolism of isocitrate was studied in this study. The results suggested that the core residues involved in isocitrate catalytic cleavage mechanism are Asp108, Cys191 and Arg228. A water molecule bonded to Mg2+ acts as the catalytic base for the deprotonation of isocitrate C(2)–OH group, while Cys191 acts as the catalytic acid. Our observation suggests that the shuttle proton from isocitrate hydroxyl group C(2) atom is favourably transferred to Asp108 instead of Glu182 with a lower activation energy of 6.2 kcal/mol. Natural bond analysis also demonstrated that pathway I involving the transfer of proton to Asp108 has a higher intermolecular interaction and charge transfer that were associated with higher stabilization energy. The QM/MM transition state stepwise catalytic mechanism of ICL agrees with the in vitro enzymatic assay whereby Asp108Ala and Cys191Ser ICL mutants lost their isocitrate cleavage activities.

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Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease

Nature Communications ◽

10.1038/s41467-021-23751-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Haixia Su ◽

Sheng Yao ◽

Wenfeng Zhao ◽

Yumin Zhang ◽

Jia Liu ◽

...

Keyword(s):

Theoretical Calculations ◽

Cysteine Proteinase ◽

Covalent Binding ◽

Food Sources ◽

Binding Mechanism ◽

Covalent Inhibitors ◽

Catalytic Cysteine ◽

3Cl Protease ◽

Covalent Ligands ◽

Covalent Inhibitor

AbstractThe ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CLpro) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CLpro. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CLpros, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.

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The structure and mechanism of action of papain

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1970.0023 ◽

1970 ◽

Vol 257 (813) ◽

pp. 237-248 ◽

Cited By ~ 61

Keyword(s):

Mechanism Of Action ◽

Catalytic Mechanism ◽

Proteolytic Enzymes ◽

Cysteine Proteinase ◽

Preceding Paper ◽

Cysteine Residue ◽

Enzymic Activity ◽

Critical Survey ◽

Cysteine Proteinases ◽

Stem Bromelain

The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

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Crystal Structure of the Mycobacterium tuberculosis dUTPase: Insights into the Catalytic Mechanism

Journal of Molecular Biology ◽

10.1016/j.jmb.2004.06.028 ◽

2004 ◽

Vol 341 (2) ◽

pp. 503-517 ◽

Cited By ~ 60

Author(s):

Sum Chan ◽

Brent Segelke ◽

Timothy Lekin ◽

Heike Krupka ◽

Uhn Soo Cho ◽

...

Keyword(s):

Crystal Structure ◽

Mycobacterium Tuberculosis ◽

Catalytic Mechanism

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Structure-Based Drug Design and Characterization of Sulfonyl-Piperazine Benzothiazinone Inhibitors of DprE1 from Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00681-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 13

Author(s):

Jérémie Piton ◽

Anthony Vocat ◽

Andréanne Lupien ◽

Caroline S. Foo ◽

Olga Riabova ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Design ◽

Antitubercular Activity ◽

Cysteine Residue ◽

Drug Candidate ◽

Structure Based Drug Design ◽

Content Type ◽

Active Site Cysteine ◽

Covalent Adducts

ABSTRACT Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1, thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis. As part of the MCZ backup program, we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulfonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile, and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ, and covalent adducts with the active-site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group, no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

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Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bj20110002 ◽

2011 ◽

Vol 436 (3) ◽

pp. 729-739 ◽

Cited By ~ 23

Author(s):

Marcio V. B. Dias ◽

William C. Snee ◽

Karen M. Bromfield ◽

Richard J. Payne ◽

Satheesh K. Palaninathan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Structural Investigation ◽

Catalytic Mechanism ◽

Shikimate Pathway ◽

Structural Rearrangement ◽

Competitive Inhibitors ◽

Structural Insights ◽

Potential Drug Targets

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19–24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

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Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bj20121107 ◽

2013 ◽

Vol 450 (1) ◽

pp. 127-139 ◽

Cited By ~ 27

Author(s):

Debajyoti Dutta ◽

Sudipta Bhattacharyya ◽

Amlan Roychowdhury ◽

Rupam Biswas ◽

Amit Kumar Das

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Site ◽

Ternary Complex ◽

Catalytic Mechanism ◽

Acyl Carrier Protein ◽

Structural Data ◽

Acid Synthesis ◽

Binary Complex ◽

C Terminus ◽

Covalently Linked

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

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