Pyrophosphate-dependent Enzymes Research Articles

Triazole-Based Thiamine Analogues as Inhibitors of Thiamine Pyrophosphate-Dependent Enzymes: 1,3-Dicarboxylate for Metal Binding.

Thiamine 1 (vitamin B1) is essential for energy metabolism, and interruption of its utilization pathways is linked to various disease states. Thiamine pyrophosphate 2a (TPP, the bioactive form of 1) functions as a coenzyme of a variety of enzymes. To understand the role of vitamin B1 in these diseases, a chemical approach is to use coenzyme analogues to compete with TPP for the enzyme active site, which abolishes the coenzyme function. Exemplified by oxythiamine 3a and triazole hydroxamate 4, chemical probes require the coenzyme analogues to be membrane-permeable and of broad inhibitory activity to the enzyme family (rather than being too selective to particular TPP-dependent enzymes). In this study, using biochemical assays, we show that changing the hydroxamate metal-binding group of 4 to a 1,3-dicarboxylate moiety leads to the potent inhibition of multiple TPP-dependent enzymes. We further demonstrate that this dianionic thiamine analogue when masked in its diester form becomes membrane-permeable and can be unmasked by esterase treatment. Taken together, our inhibitors are potentially useful chemical tools to study the roles of vitamin B1, using a prodrug mechanism, to induce the effects of thiamine deficiency in cell-based assays.

Synthesis of pyrrothiamine, a novel thiamine analogue, and evaluation of derivatives as potent and selective inhibitors of pyruvate dehydrogenase.

Inhibition of thiamine pyrophosphate (TPP)-dependent enzymes with thiamine/TPP analogues that have the central thiazolium ring replaced with other rings is well established, but a limited number of central rings have been reported. We report a novel analogue, pyrrothiamine, with a central pyrrole ring. We further develop pyrrothiamine derivatives as potent and selective inhibitors of pyruvate dehydrogenase, which might have anti-cancer potential.

Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation.

Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H+ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme.

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxythiamine, a thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of thiamine pyrophosphokinase (the enzyme that converts thiamine into its active form, thiamine pyrophosphate) hypersensitizes parasites to oxythiamine by up to 1,700-fold, consistent with oxythiamine being a substrate for thiamine pyrophosphokinase and its conversion into an antimetabolite. We show that parasites overexpressing the thiamine pyrophosphate-dependent enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are up to 15-fold more resistant to oxythiamine, consistent with the antimetabolite inactivating thiamine pyrophosphate-dependent enzymes. Our studies therefore validate thiamine utilization as an antimalarial drug target and demonstrate that a single antimalarial can simultaneously target several enzymes located within distinct organelles.

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

Emerging role of thiamine therapy for prevention and treatment of early-stage diabetic nephropathy

Thiamine supplementation may prevent and reverse early-stage diabetic nephropathy. This probably occurs by correcting diabetes-linked increased clearance of thiamine, maintaining activity and expression of thiamine pyrophosphate-dependent enzymes that help counter the adverse effects of high glucose concentrations-particularly transketolase. Evidence from experimental and clinical studies suggests that metabolism and clearance of thiamine is disturbed in diabetes leading to tissue-specific thiamine deficiency in the kidney and other sites of development of vascular complications. Thiamine supplementation prevented the development of early-stage nephropathy in diabetic rats and reversed increased urinary albumin excretion in patients with type 2 diabetes and microalbuminuria in two recent clinical trials. The thiamine monophosphate prodrug, Benfotiamine, whilst preventing early-stage development of diabetic nephropathy experimentally, has failed to produce similar clinical effect. The probable explanations for this are discussed. Further definitive trials for prevention of progression of early-stage diabetic nephropathy by thiamine are now required.

The role of 2-hydroxyacyl-CoA lyase, a thiamin pyrophosphate-dependent enzyme, in the peroxisomal metabolism of 3-methyl-branched fatty acids and 2-hydroxy straight-chain fatty acids

2-Hydroxyphytanoyl-CoA lyase (abbreviated as 2-HPCL), renamed to 2-hydroxyacyl-CoA lyase (abbreviated as HACL1), is the first peroxisomal enzyme in mammals that has been found to be dependent on TPP (thiamin pyrophosphate). It was discovered in 1999, when studying alpha-oxidation of phytanic acid. HACL1 has an important role in at least two pathways: (i) the degradation of 3-methyl-branched fatty acids like phytanic acid and (ii) the shortening of 2-hydroxy long-chain fatty acids. In both cases, HACL1 catalyses the cleavage step, which involves the splitting of a carbon-carbon bond between the first and second carbon atom in a 2-hydroxyacyl-CoA intermediate leading to the production of an (n-1) aldehyde and formyl-CoA. The latter is rapidly converted into formate and subsequently to CO(2). HACL1 is a homotetramer and has a PTS (peroxisomal targeting signal) at the C-terminal side (PTS1). No deficiency of HACL1 has been described yet in human, but thiamin deficiency might affect its activity.

Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

Glycolysis in the human parasite Entamoeba histolytica is characterized by the absence of cooperative modulation and the prevalence of pyrophosphate-dependent (over ATP-dependent) enzymes. To determine the flux-control distribution of glycolysis and understand its underlying control mechanisms, a kinetic model of the pathway was constructed by using the software gepasi. The model was based on the kinetic parameters determined in the purified recombinant enzymes, and the enzyme activities, and steady-state fluxes and metabolite concentrations determined in amoebal trophozoites. The model predicted, with a high degree of accuracy, the flux and metabolite concentrations found in trophozoites, but only when the pyrophosphate concentration was held constant; at variable pyrophosphate, the model was not able to completely account for the ATP production/consumption balance, indicating the importance of the pyrophosphate homeostasis for amoebal glycolysis. Control analysis by the model revealed that hexokinase exerted the highest flux control (73%), as a result of its low cellular activity and strong AMP inhibition. 3-Phosphoglycerate mutase also exhibited significant flux control (65%) whereas the other pathway enzymes showed little or no control. The control of the ATP concentration was also mainly exerted by ATP consuming processes and 3-phosphoglycerate mutase and hexokinase (in the producing block). The model also indicated that, in order to diminish the amoebal glycolytic flux by 50%, it was required to decrease hexokinase or 3-phosphoglycerate mutase by 24% and 55%, respectively, or by 18% for both enzymes. By contrast, to attain the same reduction in flux by inhibiting the pyrophosphate-dependent enzymes pyrophosphate-phosphofructokinase and pyruvate phosphate dikinase, they should be decreased > 70%. On the basis of metabolic control analysis, steps whose inhibition would have stronger negative effects on the energy metabolism of this parasite were identified, thus becoming alternative targets for drug design.

Pyruvate-Phosphate Dikinase of Oxymonads and Parabasalia and the Evolution of Pyrophosphate-Dependent Glycolysis in Anaerobic Eukaryotes

In pyrophosphate-dependent glycolysis, the ATP/ADP-dependent enzymes phosphofructokinase (PFK) and pyruvate kinase are replaced by the pyrophosphate-dependent PFK and pyruvate phosphate dikinase (PPDK), respectively. This variant of glycolysis is widespread among bacteria, but it also occurs in a few parasitic anaerobic eukaryotes such as Giardia and Entamoeba spp. We sequenced two genes for PPDK from the amitochondriate oxymonad Streblomastix strix and found evidence for PPDK in Trichomonas vaginalis and other parabasalia, where this enzyme was thought to be absent. The Streblomastix and Giardia genes may be related to one another, but those of Entamoeba and perhaps Trichomonas are distinct and more closely related to bacterial homologues. These findings suggest that pyrophosphate-dependent glycolysis is more widespread in eukaryotes than previously thought, enzymes from the pathway coexists with ATP-dependent more often than previously thought and may be spread by lateral transfer of genes for pyrophosphate-dependent enzymes from bacteria.

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Calpha-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.

Sequencing, cloning, and high-level expression of the pfp gene, encoding a PP(i)-dependent phosphofructokinase from the extremely thermophilic eubacterium Dictyoglomus thermophilum.

The sequencing, cloning, and expression of the pfp gene from Dictyoglomus thermophilum, which consists of 1,041 bp and encodes a pyrophosphate-dependent phosphofructokinase, are described. A phylogenetic analysis indicates that the enzyme is closely related to the pyrophosphate-dependent enzyme from Thermoproteus tenax. The recombinant and native enzymes share a high degree of similarity for most properties examined.

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during alpha-oxidation of 3-methyl-branched fatty acids.

In the third step of the alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg(2+) and thiamine pyrophosphate, a hitherto unrecognized cofactor of alpha-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon-carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant.

GroEL/GroES-dependent Reconstitution of α2β2 Tetramers of Human Mitochondrial Branched Chain α-Ketoacid Decarboxylase: OBLIGATORY INTERACTION OF CHAPERONINS WITH AN αβ DIMERIC INTERMEDIATE

The decarboxylase component (E1) of the human mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex (approximately 4-5 x 10(3) kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa alpha subunits and two 37.8-kDa beta subunits. In the present study, His6-tagged E1 alpha2 beta2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 degrees C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 M-1 s-1. Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-alpha and GroEL-beta complexes as combined substrates. However, the beta subunit was markedly more prone to misfolding than the alpha in the absence of GroEL. The alpha subunit was released as soluble monomers from the GroEL-alpha complex alone in the presence of GroES and Mg-ATP. In contrast, the beta subunit discharged from the GroEL-beta complex readily rebound to GroEL when the alpha subunit was absent. Analysis of the assembly state showed that the His6-alpha and beta subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently dimerized to produce the active alpha2 beta2 tetrameter. The purified alpha beta dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-alpha beta ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138 M-1 s-1. Dimers were regenerated from the GroEL-alpha beta complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete. Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 alpha2 beta2 tetramers.

Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate.

An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei must have evolved from an ancestral pyrophosphate-dependent enzyme.

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. A phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the change in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary history, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs.

Pyrophosphate-Dependent Phosphofructokinase from Giardia lamblia: Purification and Characterization

The pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) from Giardia lamblia has been purified to homogeneity using two methods. The purified enzyme is shown to be homogenous by its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single peak on reverse phase HPLC, The purified enzyme is a monomeric protein of M r 64 kDa as determined by SDS-PAGE and native PAGE. The enzyme fractionated as a 67-kDa protein by HPLC gel filtration. The pH optima in either the glycolytic or the gluconeogenic direction is near neutral, which is unlike any of the protozoan or bacterial enzymes, The enzyme displayed typical Michaelis-Menten kinetics. The K m values for pyrophosphate and fructose B-phosphate were 0.039 ± 0.005 and 0.25 ± 0.0196 mM, respectively. The initial velocity for the reverse reaction was also found to be hyperbolic, Different nucleoside triphosphates, including ATP, could not substitute for pyrophosphate as the phosphoryl donor. Similar to the pyrophosphate-dependent enzyme from other anaerobic protozoans and bacteria, the Giardial enzyme was not activated by fructose 2,6-bisphosphate, although this metabolite is a competitive inhibitor with respect to fructose 1,6-bisphosphate in the reverse reaction. The pH optima and the molecular weight properties of the Giardial enzyme are different from those of the two classes of the pyrophosphate dependent phosphofructokinases.

Accumulation of amyloid precursor protein-like immunoreactivity in rat brain in response to thiamine deficiency

Thiamine deficiency (TD) is a classical model of impaired cerebral oxidation. As in Alzheimer's disease (AD), TD is characterized by selective neuronal loss, decreased activities of thiamine pyrophosphate-dependent enzymes, cholinergic deficits and memory loss. Amyloid β-protein (Aβ), a ∼4 kDa fragment of the β-amyloid precursor protein (APP), accumulates in the brains of patients with AD or Down's syndrome. In the current study, we examined APP and Aβ immunoreactivity in the brains of thiamine-deficient rats. Animals received thiamine-deficient diet and libitum and daily injections of the thiamine antagonist, pyrithiamine. Immunocytochemical staining and immunoblotting utilized a rabbit polyclonal antiserum against human APP 645–694 (numbering according to APP 695 isoform). Three, 6 and 9 days of TD did not appear to damage any brain region nor change APP-like immunoreactivity. However, 13 days of TD led to pathological lesions mainly in the thalamus, mammillary body, inferior colliculus and some periventricular areas. While immunocytochemistry and thioflavine S histochemistry failed to show fibrillar β-amyloid, APP-like immunoreactivity accumulated in aggregates of swollen, abnormal neurites and perikarya along the periphery of the infarct-like lesion in the thalamus and medial geniculate nucleus. Immunoblotting of the thalamic region around the lesion revealed increased APP-like holoprotein immunoreactivity. APP-like immunoreactive neurites were scattered in the mammillary body and medial vestibular nuclei where the lesion did not resemble infarcts. In the inferior colliculus, increased perikaryal APP-like immunostaining occurred in neurons surrounding necrotic areas. Regions without apparent pathological lesions showed no alteration in APP-like immunoreactivity. Thus, the oxidative insult associated with cell loss, hemorrhage and infarct-like lesions during TD leads to altered APP metabolism. This is the first report to show a relationship between changes in APP expression, oxidative metabolism and selective cell damage caused by nutritional/cofactor deficiency. This model appears useful in defining the role of APP in the response to central nervous system injury, and may also be relevant to the pathophysiology of Wernicke-Korsakoff syndrome and AD.

Cerebellar alpha-ketoglutarate dehydrogenase activity is reduced in spinocerebellar ataxia type 1.

We measured the activity of the thiamine pyrophosphate-dependent enzyme alpha-ketoglutarate dehydrogenase complex in postmortem brain of 12 patients with the spinocerebellar ataxia type 1 form of olivopontocerebellar atrophy. alpha-Ketoglutarate dehydrogenase complex activity measured in the absence of thiamine pyrophosphate was markedly reduced (-72%) in olivopontocerebellar atrophy cerebellar cortex. Decreased activity of this key rate-limiting Krebs cycle enzyme could compromise cerebellar energy metabolism and excitatory amino acid synthesis and thereby contribute to the brain dysfunction of olivopontocerebellar atrophy.

Primary structure of the pyruvate phosphate dikinase in Entamoeba histolytica

Although glycolysis is remarkably conserved among all organisms, parasitic protozoa without mitochondria, such as Entamoeba histolytica and Giardia lamblia, and some prokaryotes possess enzymes within this pathway that are characterized by the dependence of pyrophosphate (PPi) [1,2]. In E. histolytica and G. lamblia, one of these enzymes, the pyruvate phosphate dikinase (PPDK) functions in the direction of ATP synthesis [3,4]. It is a substitute for pyruvate kinase which is absent in these protozoa. Therefore, PPDK seems to be a suitable target of antiparasitic drugs since the enzyme is of primary importance for energy generation by the parasite and it is absent from the host. PPDK is also found in C4 plants like maize and Flaveria trinervia where it plays a role as one of the key enzymes in the photosynthetic pathway [5,6]. Here it catalyzes the synthesis of phosphoenolpyruvate (PEP) to pyruvate. Pyrophosphate-dependent enzymes like

Molecular cloning of the E1β subunit of the rat branched chain α-ketoacid dehydrogenase

The rat liver E1β subunit of the branched-chain α-ketoacid dehydrogenase complex has been cloned and sequenced. The amino acid sequence is highly conserved between rat, human and bovine forms of the protein. The β subunits of the branched-chain α-ketoacid dehydrogenase complex from rat and Pseudomonas putida, and the pyruvate dehydrogenase complex from human and Bacillus subtilis show sequence similarities which are shared with two other thiamine pyrophosphate-dependent enzymes, yeast ( Hansenula polymorpha) formaldehyde transketolase and human transketolase. These similarities suggest that the β subunits may be involved in thiamine pyrophosphate binding.