Thiamin Diphosphate-dependent Decarboxylases Research Articles

Enzymatic Stetter Reaction: Computational Study of the Reaction Mechanism of MenD

Quantum chemical calculations are used to investigate the detailed reaction mechanism of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid (SEPHCHC) synthase (also known as MenD), a thiamin diphosphate-dependent decarboxylase that catalyzes the formation of SEPHCHC from 2-ketoglutarate and isochorismate. This enzyme is involved in the menaquinone biosynthesis pathway in M. tuberculosis and is thought of as a potential drug target for anti-tuberculosis therapeutics. In addition, MenD shows promise as a biocatalyst for the synthesis of 1,4-functionalized compounds. Models of the active site are constructed on the basis of available X-ray structures, and the intermediates and transition states involved in the reaction mechanism are optimized and characterized. The calculated mechanism is in good agreement with prior kinetic studies and gives new insights into the mode of action of the enzyme. In particular, the structure and role of the tetrahedral post-decarboxylation intermediate observed in X-ray structures are discussed.

Perturbation of the monomer-monomer interfaces of the benzoylformate decarboxylase tetramer.

The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer–monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer–tetramer equilibrium and to provide an ∼450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

Comparative characterisation of thiamin diphosphate-dependent decarboxylases

Several 2-keto acid decarboxylases catalyse an acyloin condensation-like carboligase reaction beside their physiological decarboxylase activity. Although many data concerning stability and catalytic potential of these enzymes are available, a standard evaluation under similar reaction conditions is lacking. In this comprehensive survey we assemble already published data combined with new studies of three bacterial pyruvate decarboxylases, yeast pyruvate decarboxylase, benzoylformate decarboxylase from Pseudomonas putida (BFD) and the branched-chain 2-keto acid decarboxylase from Lactococcus lactis (KdcA) . The obtained results proof that the optima for activity and stability are rather similar if comparable reaction conditions are used. Although the substrate ranges of the decarboxylase reaction of the various pyruvate decarboxylases are similar as well, they differ remarkably from those of BFD and KdcA. We further show that the range of acceptable donor aldehydes for the carboligase reaction of a respective enzyme can be reliably predicted from the substrate range of decarboxylase reaction.

Structural insights of the MenD from Escherichia coli reveal ThDP affinity

MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate) synthase belongs to the superfamily of thiamin diphosphate-dependent decarboxylases, which converts isochorismate and 2-oxoglutarate to SHCHC, pyruvate, and carbon dioxide. Here, we report the first crystal structure of apo-MenD from Escherichia coli determined in tetragonal crystal form. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes. Analytical gel filtration shows that EcMenD behaves as a dimer as well as a tetramer. Circular dichroism and isothermal calorimetry results confirm EcMenD dependency on ThDP, which concomitantly helps to stabilize with better configuration.

Catalyzing separation of carbon dioxide in thiamin diphosphate-promoted decarboxylation

Thiamin diphosphate-dependent decarboxylases form addition intermediates between thiamin diphosphate (ThDP) and 2-ketoacids. Although it appears that the intermediate should react without the intervention of catalysts, evidence has clearly shown that Brønsted acid catalysis occurs through a pre-associated system. This can promote separation of carbon dioxide from the residual carbanion by protonation of the carbanion. Proteins operate through pre-association and may readily promote the separation of carbon dioxide by protonating or oxidizing the nascent carbanion. Alternatively, a nucleophilic side chain may trap carbon dioxide as an unstable hemi-carbonate. Mutagenesis experiments by others have shown that enhanced activity due to the protein in the presence of thiamin diphosphate does not depend on the presence of any one proton donor, consistent with pooled activity within the active site. This form of catalysis has not been widely recognized, but should be considered an integral aspect of enzyme-promoted decarboxylation.

Molecular Mechanism of Allosteric Substrate Activation in a Thiamine Diphosphate-dependent Decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.

Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase

Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the K m value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k cat with pyruvate as a substrate.

Structural basis of substrate specificity in thiamin diphosphate dependent decarboxylases

Structural basis of substrate specificity in thiamin diphosphate dependent decarboxylases

Intermediates and Transition States in Thiamin Diphosphate-Dependent Decarboxylases. A Kinetic and NMR Study on Wild-Type Indolepyruvate Decarboxylase and Variants Using Indolepyruvate, Benzoylformate, and Pyruvate as Substrates

The thiamin diphosphate (ThDP)-dependent enzyme indolepyruvate decarboxylase (IPDC) is involved in the biosynthetic pathway of the phytohormone 3-indoleacetic acid and catalyzes the nonoxidative decarboxylation of 3-indolepyruvate to 3-indoleacetaldehyde and carbon dioxide. The steady-state distribution of covalent ThDP intermediates of IPDC reacting with 3-indolepyruvate and the alternative substrates benzoylformate and pyruvate has been analyzed by (1)H NMR spectroscopy. For the first time, we are able to isolate and directly assign covalent intermediates of ThDP with aromatic substrates. The intermediate analysis of IPDC variants is used to infer the involvement of active site side chains and functional groups of the cofactor in distinct catalytic steps during turnover of the different substrates. As a result, three residues (glutamate 468, aspartate 29, and histidine 115) positioned perpendicular to the thiazolium moiety of ThDP are involved in binding of all substrates and decarboxylation of the respective tetrahedral ThDP-substrate adducts. Most likely, interactions of these side chains with the substrate-derived carboxylate account for an optimal orientation of the substrate and/or intermediate in the course of carbon-carbon ligation and decarboxylation supporting the suggested least-motion, maximum overlap mechanism. The active site residue glutamine 383, which is located at the opposite site of the thiazolium nucleus as the "carboxylate pocket" (formed by the Glu-Asp-His triad), is central to the substrate specificity of IPDC, probably through orbital alignment. The Glu51-cofactor proton shuttle is, conjointly with the Glu-Asp-His triad, involved in multiple proton transfer steps, including ylide generation, substrate binding, and product release. Studies with para-substituted benzoylformate substrates demonstrate that the electronic properties of the substrate affect the stabilization or destabilization of the carbanion intermediate or carbanion-like transition state and in that way alter the rate dependence on decarboxylation. In conclusion, general mechanistic principles of catalysis of ThDP-dependent enzymes are discussed.

Remarkable stabilization of zwitterionic intermediates may account for a billion-fold rate acceleration by thiamin diphosphate-dependent decarboxylases.

When the E91D variant of apo-yeast pyruvate decarboxylase (EC 4.1.1. 1) is exposed to C2alpha-hydroxybenzylthiamin diphosphate, this putative intermediate is partitioned on the enzyme between release of the benzaldehyde product (as evidenced by regeneration of active enzyme) and dissociation of the proton at C2alpha to form the enamine-C2alpha-carbanion intermediate. While the pKa (the negative log of the acid dissociation constant) for this dissociation is approximately 15.4 in water, formation of the enamine at pH 6.0 on the enzyme indicates a >9 unit pKa suppression by the enzyme environment. The dramatic stabilization of this zwitterionic enamine intermediate at the active center is sufficient to account for as much as a 10(9)-fold rate acceleration on the enzyme. This "solvent" effect could be useful for achieving the bulk of the rate acceleration provided by the protein over and above that afforded by the coenzyme on all thiamin diphosphate-dependent 2-oxo acid decarboxylases.

2-Oxo-3-alkynoic Acids, Universal Mechanism-Based Inactivators of Thiamin Diphosphate-Dependent Decarboxylases: Synthesis and Evidence for Potent Inactivation of the Pyruvate Dehydrogenase Multienzyme Complex

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.