Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

Jordan, Frank; Nemeria, Natalia S.

doi:10.1016/j.bioorg.2014.08.002

Cited by 23 publications

(39 citation statements)

References 82 publications

(142 reference statements)

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“…28 This CD band was assigned to 1′,4′-iminophosphinolactylThDP, a reporter of the tetrahedral predecarboxylation intermediate on ThDP enzymes (LThDP in Scheme 2). 40 …”

Section: Resultsmentioning

confidence: 99%

Competence of Thiamin Diphosphate-Dependent Enzymes with 2′-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin

et al. 2016

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Bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine), a natural product isolated from some bacteria, has been implicated as an inhibitor of bacterial and yeast growth, as well as in inhibition of thiamin biosynthesis. Given that thiamin biosynthetic enzymes could convert bacimethrin to 2′-methoxythiamin diphosphate (MeOThDP), it is important to evaluate the effect of this coenzyme analogue on thiamin diphosphate (ThDP)-dependent enzymes. The potential functions of MeOThDP were explored on five ThDP-dependent enzymes: the human and Escherichia coli pyruvate dehydrogenase complexes (PDHc-h and PDHc-ec, respectively), the E. coli 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), and the human and E. coli 2-oxoglutarate dehydrogenase complexes (OGDHc-h and OGDHc-ec, respectively). Using several mechanistic tools (fluorescence, circular dichroism, kinetics, and mass spectrometry), it was demonstrated that MeOThDP binds in the active centers of ThDP-dependent enzymes, however, with a binding mode different from that of ThDP. While modest activities resulted from addition of MeOThDP to E. coli PDHc (6–11%) and DXPS (9–14%), suggesting that MeOThDP-derived covalent intermediates are converted to the corresponding products (albeit with rates slower than that with ThDP), remarkably strong activity (up to 75%) resulted upon addition of the coenzyme analogue to PDHc-h. With PDHc-ec and PDHc-h, the coenzyme analogue could support all reactions, including communication between components in the complex. No functional substitution of MeOThDP for ThDP was in evidence with either OGDH-h or OGDH-ec, shown to be due to tight binding of ThDP.

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“…28 This CD band was assigned to 1′,4′-iminophosphinolactylThDP, a reporter of the tetrahedral predecarboxylation intermediate on ThDP enzymes (LThDP in Scheme 2). 40 …”

Section: Resultsmentioning

confidence: 99%

Competence of Thiamin Diphosphate-Dependent Enzymes with 2′-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin

et al. 2016

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“…Cofactor activation in ThDP-dependent enzymes results from a protein environment that raises the pK a of the AP ring, favoring formation of the pyrimidinium (APH + ) and imino (IP) forms of the AP moiety (Frank et al, 2007;Jordan and Nemeria, 2014). In almost all such enzymes this depends on an active-site Glu residue, although in at least one case, glyoxalate carboligase, in which the Glu is replaced with a Val, this is achieved through other protein interactions (Kaplun et al, 2008;Nemeria et al, 2012).…”

Section: Thdp Bindingmentioning

confidence: 98%

“…In addition to their common structural organization, ThDPdependent enzymes share a general mechanism that can be described in terms of two sequential half-reactions that lead to formation of metastable intermediates (Frank et al, 2007;Jiang et al, 2007a;Jordan and Nemeria, 2014). In the first half-reaction, the thiazolium C2 carbanion attacks the first substrate to form a metastable a-carbanion/enamine intermediate I.…”

Section: Introductionmentioning

confidence: 98%

“…Despite wide sequence divergence and diverse reactions, ThDP-dependent enzymes share a conserved fold (Duggleby, 2006;Frank et al, 2007), in which paired active sites, $25 Å apart, are formed in the interface between two subunits; the enzymes are typically obligate dimers but some associate further into higher oligomers. Activation of ThDP depends on the binding of its 4 0 -aminopyrimidine (AP) ring in an environment that enables it to access catalytically relevant imino (IP) and pyrimidinium (APH + ) tautomers (Nemeria et al, 2007;Balakrishnan et al, 2012;Jordan and Nemeria, 2014) (Figure 1), and the adoption of a V-shaped conformation when the two cofactor rings are bent over a conserved hydrophobic residue (Frank et al, 2007). This brings the exocyclic N4 0 of the AP ring close to the C2 atom of the thiazolium (TA) ring, drastically lowering the pK a of this C-H moiety to facilitate its deprotonation and formation of a highly nucleophilic ylide species.…”

Section: Introductionmentioning

confidence: 99%

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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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“…Although direct spectroscopic evidence of the APH + state remains elusive, its existence has been inferred from alternative experiments (e.g., pH rate profiles, solid-state NMR) and hypothesized to assist in promoting IP formation via stabilization of the tautomerization reaction. 52,53,55 An elevation in the p K a of TDP’s N1 atom is proposed to account for the APH + state’s existence, which is justified by its proximity to a strictly conserved glutamate residue. This idea, however, does not consider the possibility of an accompanying elevation in the p K a of the glutamate residue.…”

Section: Introductionmentioning

confidence: 99%

Thiamin Diphosphate Activation in 1-Deoxy-d-xylulose 5-Phosphate Synthase: Insights into the Mechanism and Underlying Intermolecular Interactions

et al. 2016

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1-Deoxy-D-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4′-amino-pyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4′-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ~10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.

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Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

Cited by 23 publications

References 82 publications

Competence of Thiamin Diphosphate-Dependent Enzymes with 2′-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin

Competence of Thiamin Diphosphate-Dependent Enzymes with 2′-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin

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

Thiamin Diphosphate Activation in 1-Deoxy-d-xylulose 5-Phosphate Synthase: Insights into the Mechanism and Underlying Intermolecular Interactions

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