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.

The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1–4), an inactivation reversed by the action of two phosphatases (PDP1 and -2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2·E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (L1 and L2 on E2 and L3 on E3BP) lend themselves to NMR studies and determination of interaction maps with PDK1 and PDK2 at the individual residue level, HDX-MS allowed studies of interaction loci on both partners in the complexes, PDKs, and other regions of the E2·E3BP core, as well, at the peptide level. HDX-MS suggested that the intact E2·E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2·E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence of greater plasticity of PDK1 than of PDK2. The effect of L1L2S paralleled HDX-MS results obtained with the intact E2·E3BP core; hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S′ induced moderate interaction with both PDKs according to both methods.

Identification of enzyme-bound intermediates via their spectroscopic signatures, which then allows direct monitoring of the kinetic fate of these intermediates, poses a continuing challenge. As an electrophilic covalent catalyst, the thiamin diphosphate (ThDP) coenzyme forms a number of noncovalent and covalent intermediates along its reaction pathways, and multiple UV–vis and circular dichroism (CD) bands have been identified at Rutgers pertinent to several among them. These electronic transitions fall into two classes: those for which the conjugated system provides a reasonable guide to the observed λmax and others in which there is no corresponding conjugated system and the observed CD bands are best ascribed to charge transfer (CT) transitions. Herein is reported the reaction of four ThDP enzymes with alternate substrates: (a) acetyl pyruvate, its methyl ester, and fluoropyruvate, these providing the shortest side chains attached at the thiazolium C2 atom and leading to CT bands with λmax values of >390 nm, not pertinent to any on-pathway conjugated systems (estimated λmax values of <330 nm), and (b) (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid displaying both a conjugated enamine (430 nm) and a CT transition (480 nm). We suggest that the CT transitions result from an interaction of the π bond on the ThDP C2 side chain as a donor, and the positively charged thiazolium ring as an acceptor, and correspond to covalent ThDP-bound intermediates. Time resolution of these bands allows the rate constants for individual steps to be determined. These CD methods can be applied to the entire ThDP superfamily of enzymes and should find applications with other enzymes.

Thiamin diphosphate (ThDP), the vitamin B1 coenzyme is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/carbene/C2-carbanion of the thiazolium ring and the C2α-carbanion/enamine, once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of several intermediates which are stable under acidic conditions, solid-state NMR and circular dichroism detection of the states of ionization and tautomerization of the 4′-aminopyrimidine moiety of ThDP in some of the intermediates. These methods also enabled in some cases determination of the rate-limiting step in the complex series of steps. This review is an update of a review with the same title published by the authors in 2005 in this Journal. Much progress has been made in the intervening decade in the identification of the intermediates and their application to gain additional mechanistic insight.

This review is focused on three types of enzymes decarboxylating very different substrates: (1) thiamin diphosphate (ThDP)-dependent enzymes reacting with 2-oxo acids; (2) pyridoxal phosphate (PLP)-dependent enzymes reacting with α-amino acids; and (3) an enzyme with no known cofactors, orotidine 5′-monophosphate decarboxylase (OMPDC). Although the first two classes have been much studied for many years, during the past decade, studies of both classes have revealed novel mechanistic insight challenging accepted understanding. The enzyme OMPDC has posed a challenge to the enzymologist attempting to explain a 1017-fold rate acceleration in the absence of cofactors or even metal ions. A comparison of the available evidence on the three types of decarboxylases underlines some common features and more differences. The field of decarboxylases remains an interesting and challenging one for the mechanistic enzymologist, notwithstanding the large amount of information already available.Keywords: 2-oxo acids; catalysis; circular dichroism; decarboxylation; orotidine 5′-monophosphate; pyridoxal 5-phosphate; thiamin diphosphate; α-amino acids

The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-d-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of d-GAP, while addition of d-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and d-GAP bound, and the central enzyme-bound enamine reacts with d-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of d-GAP suggests a new approach to inhibitor design.

Spectroscopic identification and characterization of covalent and noncovalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. The Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), consisting of multiple copies of enzymic components and coenzymes, performs the oxidative decarboxylation of pyruvate to acetyl-CoA and is central to carbon metabolism linking glycolysis to the Krebs cycle. On the basis of earlier studies, we hypothesized that the dynamic regions of the E1p component, which undergo a disorder–order transition upon substrate binding to thiamin diphosphate (ThDP), play a critical role in modulation of the catalytic cycle of PDHc. To test our hypothesis, we kinetically characterized ThDP-bound covalent intermediates on the E1p component, and the lipoamide-bound covalent intermediate on the E2p component in PDHc and in its variants with disrupted active-site loops. Our results suggest that formation of the first covalent predecarboxylation intermediate, C2α-lactylthiamin diphosphate (LThDP), is rate limiting for the series of steps culminating in acetyl-CoA formation. Substitutions in the active center loops produced variants with up to 900-fold lower rates of formation of the LThDP, demonstrating that these perturbations directly affected covalent catalysis. This rate was rescued by up to 5-fold upon assembly to PDHc of the E401K variant. The E1p loop dynamics control covalent catalysis with ThDP and are modulated by PDHc assembly, presumably by selection of catalytically competent loop conformations. This mechanism could be a general feature of 2-oxoacid dehydrogenase complexes because such interfacial dynamic regions are highly conserved.

The bacterial pyruvate dehydrogenase complex carries out conversion of pyruvate to acetyl-coenzyme A with the assistance of thiamin diphosphate (ThDP), several other cofactors, and three principal protein components, E1–E3, each present in multiple copies. The E2 component forms the core of the complexes, each copy consisting of variable numbers of lipoyl domains (LDs, lipoic acid covalently amidated at a lysine residue), peripheral subunit binding domains (PSBDs), and catalytic (or core) domains (CDs). The reaction starts with a ThDP-dependent decarboxylation on E1 to an enamine/C2α̃ carbanion, followed by oxidation and acetyl transfer to form S-acetyldihydrolipoamide E2, and then transfer of this acetyl group from the LD to coenzyme A on the CD. The dihydrolipoamide E2 is finally reoxidized by the E3 component. This report investigates whether the acetyl group is passed from the LD to the CD in an intra- or interchain reaction. Using an Escherichia coli E2 component having a single LD, two types of constructs were prepared: one with a Lys to Ala substitution in the LD at the Lys carrying the lipoic acid, making E2 incompetent toward post-translational ligation of lipoic acid and, hence, toward reductive acetylation, and the other in which the His believed to catalyze the transthiolacetylation in the CD is substituted with A or C, the absence of His rendering it incompetent toward acetyl-CoA formation. Both kinetic evidence and mass spectrometric evidence support interchain transfer of the acetyl groups, providing a novel model for the presence of multiples of three chains in all E2 components, and their assembly in bacterial enzymes.

Knowledge of the state of ionization and tautomerization of heteroaromatic cofactors when enzyme-bound is essential for formulating a detailed stepwise mechanism via proton transfers, the most commonly observed contribution to enzyme catalysis. In the bifunctional coenzyme, thiamin diphosphate (ThDP), both aromatic rings participate in catalysis, the thiazolium ring as an electrophilic covalent catalyst and the 4′-aminopyrimidine as acid–base catalyst involving its 1′,4′-iminopyrimidine tautomeric form. Two of four ionization and tautomeric states of ThDP are well characterized via circular dichroism spectral signatures on several ThDP superfamily members. Yet, the method is incapable of providing information about specific proton locations, which in principle may be accessible via NMR studies. To determine the precise ionization/tautomerization states of ThDP during various stages of the catalytic cycle, we report the first application of solid-state NMR spectroscopy to ThDP enzymes, whose large mass (160,000–250,000 Da) precludes solution NMR approaches. Three de novo synthesized analogues, [C2,C6′-13C2]ThDP, [C2-13C]ThDP, and [N4′-15N]ThDP used with three enzymes revealed that (a) binding to the enzymes activates both the 4′-aminopyrimidine (via pKa elevation) and the thiazolium rings (pKa suppression); (b) detection of a pre-decarboxylation intermediate analogue using [C2,C6′-13C2]ThDP, enables both confirmation of covalent bond formation and response in 4′-aminopyrimidine ring’s tautomeric state to intermediate formation, supporting the mechanism we postulate; and (c) the chemical shift of bound [N4′-15N]ThDP provides plausible models for the participation of the 1′,4′-iminopyrimidine tautomer in the mechanism. Unprecedented detail is achieved about proton positions on this bifunctional coenzyme on large enzymes in their active states.

The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the “gatekeeper” for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate but E2o for 2-oxoglutarate.

The region encompassing residues 401–413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541–557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: (a) sequester the active center from carboligase side reactions; (b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; (c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an 19F NMR label onto these engineered cysteines, the loop mobility was examined: (a) both methods suggested that in the absence of ligand, the loop exists in two conformations; (b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex.

•Synthesis of chiral compounds using E1 of 2-oxoglutarate dehydrogenase is reported.•Chiral 2-hydroxyketones are synthesized varying donor and acceptor substrates.•Chiral products with (R) or (S) enantiomers were produced with 60–95% ee.•Use of an ester and a 2-oxoaldehyde as acceptors for the enamine is accomplished.•2-Oxovalerate and 2-oxoisovalerate are also accepted in carboligation as donors.The potential of thiamin diphosphate (ThDP)-dependent enzymes to catalyze CC bond forming (carboligase) reactions with high enantiomeric excess has been recognized for many years. Here we report the application of the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex in the synthesis of chiral compounds with multiple functional groups in good yield and high enantiomeric excess, by varying both the donor substrate (different 2-oxo acids) and the acceptor substrate (glyoxylate, ethyl glyoxylate and methyl glyoxal). Major findings include the demonstration that the enzyme can accept 2-oxovalerate and 2-oxoisovalerate in addition to its natural substrate 2-oxoglutarate, and that the tested acceptors are also acceptable in the carboligation reaction, thereby very much expanding the repertory of the enzyme in chiral synthesis.Download full-size image