Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis

2015 ◽  
Vol 192 (1) ◽  
pp. 76-87 ◽  
Author(s):  
Andrew C. Pratt ◽  
Sajeewa W. Dewage ◽  
Allan H. Pang ◽  
Tapan Biswas ◽  
Sandra Barnard-Britson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Catalytic Mechanism ◽  
Inorganic Pyrophosphatase ◽  
Computational Dissection

Design of a novel Peltier-based cooling device and its use in neutron diffraction data collection of perdeuterated yeast pyrophosphatase

2010 ◽  
Vol 43 (5) ◽  
pp. 1113-1120 ◽  
Author(s):  
Esko Oksanen ◽  
François Dauvergne ◽  
Adrian Goldman ◽  
Monika Budayova-Spano
Keyword(s):  
Data Collection ◽  
Neutron Diffraction ◽  
Diffraction Data ◽  
Catalytic Mechanism ◽  
Inorganic Pyrophosphatase ◽  
Neutron Diffraction Data ◽  
Cooling Device ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography

H atoms play a central role in enzymatic mechanisms, but H-atom positions cannot generally be determined by X-ray crystallography. Neutron crystallography, on the other hand, can be used to determine H-atom positions but it is experimentally very challenging. Yeast inorganic pyrophosphatase (PPase) is an essential enzyme that has been studied extensively by X-ray crystallography, yet the details of the catalytic mechanism remain incompletely understood. The temperature instability of PPase crystals has in the past prevented the collection of a neutron diffraction data set. This paper reports how the crystal growth has been optimized in temperature-controlled conditions. To stabilize the crystals during neutron data collection a Peltier cooling device that minimizes the temperature gradient along the capillary has been developed. This device allowed the collection of a full neutron diffraction data set.

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

2004 ◽  
Vol 341 (2) ◽  
pp. 503-517 ◽  
Author(s):  
Sum Chan ◽  
Brent Segelke ◽  
Timothy Lekin ◽  
Heike Krupka ◽  
Uhn Soo Cho ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Catalytic Mechanism

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

2011 ◽  
Vol 436 (3) ◽  
pp. 729-739 ◽  
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.

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

2013 ◽  
Vol 450 (1) ◽  
pp. 127-139 ◽  
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.

Structural insights of catalytic mechanism in mutant pyrazinamidase of Mycobacterium tuberculosis

2020 ◽  
pp. 1-14
Author(s):  
Muhammad Junaid ◽  
Cheng-Dong Li ◽  
Jiayi Li ◽  
Abbas Khan ◽  
Syed Shujait Ali ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Catalytic Mechanism ◽  
Structural Insights

scholarly journals Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis isocitrate lyase

2020 ◽  
Author(s):  
Brooke X. C. Kwai ◽  
Annabelle J. Collins ◽  
Martin J. Middleditch ◽  
Jonathan Sperry ◽  
Ghader Bashiri ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Catalytic Mechanism ◽  
Isocitrate Lyase ◽  
Cysteine Residue ◽  
Covalent Adduct ◽  
Catalytic Cysteine ◽  
Covalent Inhibitor

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.

scholarly journals Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

2011 ◽  
Vol 286 (18) ◽  
pp. 16197-16207 ◽  
Author(s):  
Sebastian Reichau ◽  
Wanting Jiao ◽  
Scott R. Walker ◽  
Richard D. Hutton ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Condensation Reaction ◽  
Shikimate Pathway ◽  
Enzyme Reaction ◽  
Multidrug Resistant ◽  
Active Site Residues ◽  
Site Water

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

scholarly journals Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C

2002 ◽  
Vol 367 (1) ◽  
pp. 255-261 ◽  
Author(s):  
Radha CHAUHAN ◽  
Shekhar C. MANDE
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reaction Mechanism ◽  
Kinetic Parameters ◽  
Active Site ◽  
Double Mutant ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Resistant Strains

Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.

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