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ABSTRACT: Thiamin diphosphate (ThDP)-dependent enzymes play vital roles in cellular metabolism in all kingdoms of life. In previous kinetic and structural studies, a communication between the active centers in terms of a negative cooperativity had been suggested for some but not all ThDP enzymes, which typically operate as functional dimers. To further underline this hypothesis and to test its universality, we investigated the binding of substrate analogue methyl acetylphosphonate (MAP) to three different ThDP-dependent enzymes acting on substrate pyruvate, namely, the Escherichia coli E1 component of the pyruvate dehydrogenase complex, E. coli acetohydroxyacid synthase isoenzyme I, and the Lactobacillus plantarum pyruvate oxidase using isothermal titration calorimetry. The results unambiguously show for all three enzymes studied that only one active center of the functional dimers accomplishes covalent binding of the substrate analogue, supporting the proposed alternating sites reactivity as a common feature of all ThDP enzymes and resolving the recent controversy in the field.
Biochemistry 04/2013; · 3.42 Impact Factor
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ABSTRACT: Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.
Proceedings of the National Academy of Sciences 06/2012; 109(27):10867-72. · 9.68 Impact Factor
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ABSTRACT: Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a "pseudo-TKTL1" Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.
PLoS ONE 01/2012; 7(10):e48321. · 4.09 Impact Factor
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ABSTRACT: Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the first common step in the biosynthesis of branched-chain amino acids, condensation of pyruvate with a second 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme II from Escherichia coli is specific for pyruvate as the first donor substrate but exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. In previous studies relying on steady state and transient kinetics, substrate competition and detailed analysis of the distribution of intermediates in the steady-state, we have identified several residues which confer specificity for the donor and acceptor substrates, respectively. Here, we examine the roles of active site polar residues Glu47, Gln110, Lys159, and His251 for elementary steps of catalysis using similar approaches. While Glu47, the conserved essential glutamate conserved in all ThDP-dependent enzymes whose carboxylate is in H-bonding distance of the ThDP iminopyrimidine N1', is involved as expected in cofactor activation, substrate binding, and product elimination, our studies further suggest a crucial catalytic role for it in the carboligation of the acceptor and the hydroxyethyl-ThDP enamine intermediate. The Glu47-cofactor proton shuttle acts in concert with Gln110 in the carboligation. We suggest that either the transient oxyanion on the acceptor carbonyl is stabilized by H-bonding to the glutamine side chain, or carboligation involves glutamine tautomerization and the elementary reactions of addition and protonation occur in a concerted manner. This is in contrast to the situation in other ThDP enzymes that catalyze a carboligation, such as, e.g., transketolase or benzaldehyde lyase, where histidines act as general acid/base catalysts. Our studies further suggest global catalytic roles for Gln110 and Glu47, which are engaged in all major bond-breaking and bond-making steps. In contrast to earlier suggestions, Lys159 has a minor effect on the kinetics and specificity of AHAS II, far less than does Arg276, previously shown to influence the specificity for a 2-ketoacid as a second substrate. His251 has a large effect on donor substrate binding, but this effect masks any other effects of replacement of His251.
Biochemistry 03/2011; 50(15):3250-60. · 3.42 Impact Factor
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ABSTRACT: Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate into acetaldehyde and CO(2) and requires the cofactors thiamin diphosphate and Mg(2+) for activity. Owing to its catalytic promiscuity and relaxed substrate specificity, PDC catalyzes carboligation side reactions and is exploited for the asymmetric synthesis of 2-hydroxy ketones such as (R)-phenylacetyl carbinol, the precursor of (-)-ephedrine. Although PDC variants with enhanced carboligation efficiency were generated in the past, the native reaction, i.e., formation of aldehydes, is heavily favored over carboligation side reactions in all these biocatalysts. We characterized an active site variant (Glu473Gln) in which partitioning between aldehyde release versus carboligation is inverted with an up to 100-fold preference for the latter pathway. Due to a defective protonation of the central carbanion/enamine intermediate, substrate turnover stalls at this catalytic stage and addition of external aldehydes leads to quantitative and enantioselective formation of 2-hydroxy ketones as shown for (R)-phenylacetyl carbinol, which is afforded with unmatched yields, rates, and purity. This protein variant thus constitutes an example for the rational design of biocatalysts with greatly enhanced accidental catalytic promiscuity by selective blockage of the native reaction and accumulation of reactive intermediates under steady-state turnover conditions.
Journal of the American Chemical Society 02/2011; 133(10):3609-16. · 9.91 Impact Factor
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ABSTRACT: Pyruvate decarboxylase (PDC) catalyzes the nonoxidative decarboxylation of pyruvate into acetaldehyde and carbon dioxide and requires thiamin diphosphate (ThDP) and a divalent cation as cofactors. Recent studies have permitted the assignment of functional roles of active site residues; however, the underlying reaction mechanisms of elementary steps have remained hypothetical. Here, a kinetic and thermodynamic single-step analysis in conjunction with X-ray crystallographic studies of PDC from Zymomonas mobilis implicates active site residue Glu473 (located on the re-face of the ThDP thiazolium nucleus) in facilitating both decarboxylation of 2-lactyl-ThDP and protonation of the 2-hydroxyethyl-ThDP carbanion/enamine intermediate. Variants carrying either an isofunctional (Glu473Asp) or isosteric (Glu473Gln) substitution exhibit a residual catalytic activity of less than 0.1% but accumulate different intermediates at the steady state. Whereas the predecarboxylation intermediate 2-lactyl-ThDP is accumulated in Glu473Asp because of a 3000-fold slower decarboxylation compared to that of the wild-type enzyme, Glu473Gln is not impaired in decarboxylation but generates a long-lived 2-hydroxyethyl-ThDP carbanion/enamine postdecarboxylation intermediate. CD spectroscopic analysis of the protonic and tautomeric equilibria of the cocatalytic aminopyrimidine part of ThDP indicates that an acidic residue is required at position 473 for proper substrate binding. Wild-type PDC and the Glu473Asp variant bind the substrate analogue acetylphosphinate with the same affinity, implying a similar stabilization of the predecarboxylation intermediate analogue on the enzyme, whereas Glu473Gln fails to bind the analogue. The X-ray crystallographic structure of 2-lactyl-ThDP trapped in the decarboxylation-deficient variant Glu473Asp reveals a common stereochemistry of the intermediate C2α stereocenter; however, the scissile C2α-C(carboxylate) bond deviates by ∼25-30° from the perpendicular "maximum overlap" orientation relative to the thiazolium ring plane as commonly observed in ThDP enzymes. Because a reactant-state stabilization of the predecarboxylation intermediate can be excluded to account for the slower decarboxylation, the data suggest a strong stereoelectronic effect for the transition state of decarboxylation as supported by additional DFT studies on models. To the best of our knowledge, this is a very rare example in which the magnitude of a stereoelectronic effect could be experimentally estimated for an enzymatic system. Given that variant Glu473Gln is not decarboxylation-deficient, electrostatic stress can be excluded as a driving force for decarboxylation. The apparent dual function of Glu473 further suggests that decarboxylation and protonation of the incipient carbanion are committed and presumably proceed in the same transition state.
Biochemistry 09/2010; 49(37):8197-212. · 3.42 Impact Factor
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ABSTRACT: Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate-dependent enzyme that catalyzes the condensation of pyruvate with either another pyruvate molecule (product acetolactate) or 2-ketobutyrate (product acetohydroxybutyrate) as the first common step in the biosynthesis of branched-chain amino acids in plants, bacteria, algae, and fungi. AHAS isozyme II from Escherichia coli exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as acceptor, which was shown to result from a stronger hydrophobic interaction of the ethyl substituent of 2-KB with the side chain of Trp464 in multiple, apparently committed steps of catalysis. Here, we have elucidated the molecular determinants conferring specificity for pyruvate as the sole physiological donor substrate. Structural studies and sequence alignments of the POX subfamily of ThDP enzymes that act on pyruvate indicate that a valine and a phenylalanine hydrophobically interact with the methyl substituent of pyruvate. Kinetic and thermodynamic studies on AHAS isozyme II variants with substitutions at these positions (Val375Ala, Val375Ile, and Phe109Met) were carried out. While Val375 variants exhibit a slightly reduced k(cat) with a moderate increase of the apparent K(M) of pyruvate, both substrate affinity and k(cat) are significantly compromised in AHAS Phe109Met. The specificity for 2-ketobutyrate as acceptor is not altered in the variants. Binding of acylphosphonates as analogues of donor substrates was analyzed by circular dichroism spectroscopy and stopped-flow kinetics. While binding of the pyruvate analogue is 10-100-fold compromised in all variants, Val375Ala binds the 2-KB analogue better than the wild type and with higher affinity than the pyruvate analogue, suggesting steric constraints imposed by Val375 as a major determinant for the thermodynamically favored binding of pyruvate in AHAS. NMR-based intermediate analysis at steady state reveals that a mutation of either Val375 or Phe109 is detrimental for unimolecular catalytic steps in which tetrahedral intermediates are involved, such as substrate addition to the cofactor and product liberation. This observation implies Val375 and Phe109 to not only conjointly mediate substrate binding and specificity but moreover to ensure a proper orientation of the donor substrate and intermediates for correct orbital alignment in multiple transition states.
Biochemistry 06/2010; 49(25):5188-99. · 3.42 Impact Factor
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ABSTRACT: The thiamin diphosphate- (ThDP-) dependent enzyme benzoylformate decarboxylase (BFDC) catalyzes the nonoxidative decarboxylation of benzoylformic acid to benzaldehyde and carbon dioxide. To date, no structural information for a cofactor-bound reaction intermediate in BFDC is available. For kinetic analysis, a chromophoric substrate analogue was employed that produces various absorbing intermediates during turnover but is a poor substrate with a 10(4)-fold compromised kcat. Here, we have analyzed the steady-state distribution of native intermediates by a combined chemical quench/1H NMR spectroscopic approach and estimated the net rate constants of elementary catalytic steps. At substrate saturation, carbonyl addition of the substrate to the cofactor (k' approximately 500 s-1 at 30 degrees C) and elimination of benzaldehyde (k' approximately 2.400 s-1) were found to be partially rate-determining for catalysis, whereas decarboxylation of the transient 2-mandelyl-ThDP intermediate is 1 order of magnitude faster with k' approximately 16.000 s-1, the largest rate constant of decarboxylation in any thiamin enzyme characterized so far. The X-ray structure of a predecarboxylation intermediate analogue was determined to 1.6 A after cocrystallization of BFDC from Pseudomonas putida with benzoylphosphonic acid methyl ester. In contrast to the free acid, for which irreversible phosphorylation of active center Ser26 was reported, the methyl ester forms a covalent adduct with ThDP with a similar configuration at C2alpha as observed for other thiamin enzymes. The C2-C2alpha bond of the intermediate analogue is out of plane by 7degrees, indicating strain. The phosphonate part of the adduct forms hydrogen bonds with Ser26 and His281, and the 1-OH group is held in place by interactions with His70 and the 4'-amino group of ThDP. The phenyl ring accommodates in a hydrophobic pocket formed by Phe464, Phe397, Leu109, and Leu403. A comparison with the previously determined structure of BFDC in noncovalent complex with the inhibitor (R)-mandelate suggests a least motion mechanism. Binding of benzoylphosphonic acid methyl ester to BFDC was further characterized by CD spectroscopy and stopped-flow kinetics, indicating a two-step binding mechanism with a 200-fold slower carbonyl addition to ThDP than determined for benzoylformic acid, in line with the observed slight structural reorganization of Phe464 due to steric clashes with the phosphonate moiety.
Biochemistry 02/2009; 48(15):3258-68. · 3.42 Impact Factor
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ABSTRACT: Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.
Nature Chemical Biology 07/2006; 2(6):324-8. · 14.69 Impact Factor
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15th International Symposium on Flavins and Flavoproteins;
