Crystallographic Snapshots of Oxalyl-CoA Decarboxylase Give Insights into Catalysis by Nonoxidative ThDP-Dependent Decarboxylases

Berthold, C.L.; Toyota, C.G.; Moussatche, Patricia; Wood, M.; Leeper, Finian J.; Richards, Nigel G. J.; Lindqvist, Ylva

doi:10.1016/j.str.2007.06.001

Cited by 51 publications

(78 citation statements)

References 36 publications

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“…The thiazolium ring is slightly distorted such that C2a, expected to lie in the thiazolium ring plane, is about 0.3 Å out of plane, displaced in a direction away from the AP N4 0 atom. Similar out-of-plane displacement has been seen in high-resolution structures of the equivalent intermediates of other ThDP-dependent enzymes (Berthold et al, 2007;Ludtke et al, 2013). A crucial feature in the Mtb-MenD structure, however, is that the C2a-OH is tilted further away from the AP N4 0 to a distance (4.5 Å ) too great for attack, thus preventing premature product release, as described later.…”

Section: The Catalytic Mechanism Tracked Through Its Two Covalent Insupporting

confidence: 67%

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

et al. 2016

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Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.

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Section: The Catalytic Mechanism Tracked Through Its Two Covalent Insupporting

confidence: 67%

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

et al. 2016

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“…The first stage of the reaction involves the formation of a covalent adduct between the cofactor and α-ketoglutarate; the formation of such species is well established in ThDP-dependent enzymes 24 and the adduct structure has been captured crystallographically 25 . The driving force for this stage largely comes from the geometry of the cofactor in the binding site, which brings N4′ of the pyrimidine ring and C2 of the thiazolium ring into close proximity.…”

Section: Resultsmentioning

confidence: 99%

Structure and Reactivity of Bacillus subtilis MenD Catalyzing the First Committed Step in Menaquinone Biosynthesis

Dawson¹,

Chen²,

Fyfe³

et al. 2010

Journal of Molecular Biology

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The first committed step in the classical biosynthetic route to menaquinone (vitamin K2) is a Stetter-like conjugate addition of α-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 Å) crystal structure of Bacillus subtilis MenD with cofactor and Mn2+ has been determined. Based on structure–sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of α-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure–reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.

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“…It should be noted that the holo structure presented here has a water molecule at approximately this position in all four active sites. For oxalyl CoA decarboxylase, a structure of the enamine intermediate has been determined, and it has a water molecule in just such a position, poised for protonation of the enamine carbon (46). …”

Section: Discussionmentioning

confidence: 99%

Structural Insights into the Prereaction State of Pyruvate Decarboxylase from Zymomonas mobilis,

et al. 2010

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Pyruvate decarboxylase (PDC) uses thiamine diphosphate as an essential cofactor to catalyze the formation of acetaldehyde on the pathway of ethanol synthesis. Here we report the crystallographic image of a prereaction intermediate of a bacterial pyruvate decarboxylase prepared by cocrystallizing the enzyme with pyruvate and a stable analogue of the cofactor’s activated ylid form. A second crystal structure of PDC in complex with fluoride shows that the ion organizes a water molecule that occludes the pyruvate binding site, accounting for the inhibitory effect of the halide. Also reported is a structure of the cofactor-free apo form, which when compared to the structure of the holo form indicates how thiamine diphosphate organizes the active site pocket of pyruvate decarboxylase to support catalysis. Guided by the structural and enzymatic data, we propose roles for several key residues in the catalytic mechanism.

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Crystallographic Snapshots of Oxalyl-CoA Decarboxylase Give Insights into Catalysis by Nonoxidative ThDP-Dependent Decarboxylases

Cited by 51 publications

References 36 publications

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

Structure and Reactivity of Bacillus subtilis MenD Catalyzing the First Committed Step in Menaquinone Biosynthesis

Structural Insights into the Prereaction State of Pyruvate Decarboxylase from Zymomonas mobilis,

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