Structural insights of catalytic mechanism in mutant pyrazinamidase of Mycobacterium tuberculosis

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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Faculty Opinions recommendation of Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1009676.250616 ◽

2004 ◽

Author(s):

Fred Dyda

Keyword(s):

Catalytic Mechanism ◽

Ester Hydrolysis ◽

Phosphate Ester ◽

Phosphate Ester Hydrolysis ◽

Structural Insights

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Structural insights into the catalytic mechanism of sphingomyelinases D and evolutionary relationship to glycerophosphodiester phosphodiesterases

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2006.01.123 ◽

2006 ◽

Vol 342 (1) ◽

pp. 323-329 ◽

Cited By ~ 45

Author(s):

Mário T. Murakami ◽

Matheus Freitas Fernandes-Pedrosa ◽

Sonia A. de Andrade ◽

Azat Gabdoulkhakov ◽

Christian Betzel ◽

...

Keyword(s):

Catalytic Mechanism ◽

Evolutionary Relationship ◽

Structural Insights

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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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Structural insights into the specificity and catalytic mechanism of mycobacterial nucleotide pool sanitizing enzyme MutT2

Journal of Structural Biology ◽

10.1016/j.jsb.2018.10.002 ◽

2018 ◽

Vol 204 (3) ◽

pp. 449-456 ◽

Cited By ~ 1

Author(s):

Amandeep Singh ◽

Sheikh Mohammad Arif ◽

Pau Biak Sang ◽

Umesh Varshney ◽

M. Vijayan

Keyword(s):

Catalytic Mechanism ◽

Structural Insights

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Structural Insights of LspA, from Mycobacterium Tuberculosis, using Solid State NMR Spectroscopy

Biophysical Journal ◽

10.1016/j.bpj.2013.11.348 ◽

2014 ◽

Vol 106 (2) ◽

pp. 48a-49a

Author(s):

E. Vindana Ekanayake ◽

Huajun Qin ◽

Ivan Hung ◽

Timothy A. Cross

Keyword(s):

Mycobacterium Tuberculosis ◽

Solid State ◽

Nmr Spectroscopy ◽

Solid State Nmr ◽

Solid State Nmr Spectroscopy ◽

Structural Insights

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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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Structural Insights into the Molecular Evolution of the Archaeal Exo-β-d-Glucosaminidase

International Journal of Molecular Sciences ◽

10.3390/ijms20102460 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2460

Author(s):

Shouhei Mine ◽

Masahiro Watanabe

Keyword(s):

Molecular Evolution ◽

Primary Structure ◽

Active Site ◽

Catalytic Mechanism ◽

Thermostable Enzyme ◽

Crystallographic Analysis ◽

Evolutionary Pathway ◽

Chitosan Oligosaccharides ◽

Tim Barrel ◽

Structural Insights

The archaeal exo-β-d-glucosaminidase (GlmA), a thermostable enzyme belonging to the glycosidase hydrolase (GH) 35 family, hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a novel enzyme in terms of its primary structure, as it is homologous to both GH35 and GH42 β-galactosidases. The catalytic mechanism of GlmA is not known. Here, we summarize the recent reports on the crystallographic analysis of GlmA. GlmA is a homodimer, with each subunit comprising three distinct domains: a catalytic TIM-barrel domain, an α/β domain, and a β1 domain. Surprisingly, the structure of GlmA presents features common to GH35 and GH42 β-galactosidases, with the domain organization resembling that of GH42 β-galactosidases and the active-site architecture resembling that of GH35 β-galactosidases. Additionally, the GlmA structure also provides critical information about its catalytic mechanism, in particular, on how the enzyme can recognize glucosamine. Finally, we postulate an evolutionary pathway based on the structure of an ancestor GlmA to extant GH35 and GH42 β-galactosidases.

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