Proton abstraction reaction, steady-state kinetics, and oxidation-reduction potential of human glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA and CO(2) in the mitochondrial degradation of lysine, hydroxylysine, and tryptophan. We have characterized the human enzyme that was expressed in Escherichia coli. Anaerobic reduction of the enzyme with sodium dithionite or substrate yields no detectable semiquinone; however, like other acyl-CoA dehydrogenases, the human enzyme stabilizes an anionic semiquinone upon reduction of the complex between the enzyme and 2,3-enoyl-CoA product. The flavin potential of the free enzyme determined by the xanthine-xanthine oxidase method is -0.132 V at pH 7.0, slightly more negative than that of related flavoprotein dehydrogenases. A single equivalent of substrate reduces 26% of the dehydrogenase flavin, suggesting that the redox equilibrium on the enzyme between substrate and product and oxidized and reduced flavin is not as favorable as that observed with other acyl-CoA dehydrogenases. This equilibrium is, however, similar to that observed in isovaleryl-CoA dehydrogenase. Comparison of steady-state kinetic constants of glutaryl-CoA dehydrogenase with glutaryl-CoA and the alternative substrates, pentanoyl-CoA and hexanoyl-CoA, suggests that the gamma-carboxyl group of glutaryl-CoA stabilizes the enzyme-substrate complex by at least 5.7 kJ/mol, perhaps by interaction with Arg94 or Ser98. Glu370 is positioned to function as the catalytic base, and previous studies indicate that the conjugate acid of Glu370 also protonates the transient crotonyl-CoA anion following decarboxylation [Gomes, B., Fendrich, G. , and Abeles, R. H. (1981) Biochemistry 20, 3154-3160]. Glu370Asp and Glu370Gln mutants of glutaryl-CoA dehydrogenase exhibit 7% and 0. 04% residual activity, respectively, with human electron-transfer flavoprotein; these mutations do not grossly affect the flavin redox potentials of the mutant enzymes. The reduced catalytic activities of these mutants can be attributed to reduced extent and rate of substrate deprotonation based on experiments with the nonoxidizable substrate analogue, 3-thiaglutaryl-CoA, and kinetic experiments. Determination of these fundamental properties of the human enzyme will serve as the basis for future studies of the decarboxylation reaction which is unique among the acyl-CoA dehydrogenases.     

Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism

Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors.     

Pyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications

Pyruvate dehydrogenase kinase (PDK) can regulate the catalytic activity of pyruvate decarboxylation oxidation via the mitochondrial pyruvate dehydrogenase complex, and it further links glycolysis with the tricarboxylic acid cycle and ATP generation. This review seeks to elucidate the regulation of PDK activity in different species, mainly mammals, and the role of PDK inhibitors in preventing increased blood glucose, reducing injury caused by myocardial ischemia, and inducing apoptosis of tumor cells. Regulations of PDKs expression or activity represent a very promising approach for treatment of metabolic diseases including diabetes, heart failure, and cancer. The future research and development could be more focused on the biochemical understanding of the diseases, which would help understand the cellular energy metabolism and its regulation by pharmacological effectors of PDKs.     

Spectral and electrochemical properties of glutaryl-CoA dehydrogenase from Paracoccus denitrificans

Studies of the spectral (UV/vis and resonance Raman) and electrochemical properties of the FAD-containing enzyme glutaryl-CoA dehydrogenase (GCD) from Paracoccus denitrificans reveal that the properties of the oxidized enzyme (GCDox) appear to be invariant from those properties known for other acyl-CoA dehydrogenases such as mammalian general acyl-CoA dehydrogenase (GACD) and butyryl-CoA dehydrogenase (BCD) from Megasphaera elsdenii. However, when either free or complexed GCD is reduced, its spectral and electrochemical behavior differs from that of both GACD and BCD. Free GCD does not stabilize any form of one-electron-reduced GCD, but when GCD is complexed to its inhibitor, aceto-acetyl-CoA, the enzyme stabilizes 20% of the blue neutral radical form of FAD (FADH.) upon reduction. Like GACD, when crotonyl-CoA- (CCoA) bound GCD is reduced, the red anionic form of FAD radical (FAD.-) is stabilized, and excess reduction equivalents are necessary to effect full reduction of the complex. A comproportionation reaction is proposed between fully reduced crotonyl-CoA-bound GCD (GCD2e-CCoA) and GCDox-CCoA to partially explain the stabilization of GCD-bound FAD.- by CCoA. When GCD is reduced by its optimal substrate, glutaryl-CoA, a two-electron reduction is observed with concomitant formation of a long-wavelength charge-transfer band. It is proposed that the ETF specific for GCD abstracts one electron from this charge-transfer species and this is followed by the decarboxylation of the oxidized substrate. At pH 6.4, potential values measured for free GCD and GCD bound to acetoacetyl-CoA are -0.085 and -0.129 V, respectively. Experimental evidence is given for a positive shift in the reduction potential of GCD when the enzyme is bound to a 1:1 mixture of butyryl-CoA and CCoA. However, significant GCD hydratase activity is observed, preventing quantitation of the potential shift.     

Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.

Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in the oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-CoA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehydrogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity, a property of other members of the acyl-CoA dehydrogenase family. The enzyme rapidly hydrates glutaconyl-CoA at pH 7.6 with a k(cat) of 2.7 s(-1). The k(cat) in the overall oxidation-decarboxylation reaction at pH 7.6 is about 9 s(-1). The binding of glutaconyl-CoA was quantitatively assessed from the K(m) in the hydratase reaction, 3 microM, and the K(i), 1.0 microM, as a competitive inhibitor of the dehydrogenase. These values compare with K(m) and K(i) of 4.0 and 12.9 microM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an alpha-proton of the substrate to initiate the catalytic pathway. The mutant dehydrogenase, Glu370Gln, is inactive in the dehydrogenation and the hydratase reactions. However, this mutant dehydrogenase decarboxylates glutaconyl-CoA to crotonyl-CoA without oxidation-reduction reactions of the dehydrogenase flavin. Addition of glutaconyl-CoA to this mutant dehydrogenase results in a rapid, transient increase in long-wavelength absorbance (lambda(max) approximately 725 nm), and crotonyl-CoA is found as the sole product. We propose that this 725 nm-absorbing species is the delocalized crotonyl-CoA anion that follows decarboxylation and that the decay is the result of slow protonation of the anion in the absence of the general acid catalyst, Glu370(H(+)). In the absence of detectable oxidation-reduction, the data indicate that oxidation-reduction of the dehydrogenase flavin is not essential for decarboxylation of glutaconyl-CoA.     

Kinetic and structural evidence for the identification of 11-hydroxythromboxane B2 dehydrogenase as cytosolic aldehyde dehydrogenase

We have recently purified 11-hydroxythromboxane B₂ dehydrogenase from porcine kidney and identified it as cytosolic aldehyde dehydrogenase (EC 1.2.1.3) based on amino acid analysis and other protein characteristics. In the present paper we have studied the catalytic interaction of thromboxane B₂ (TXB₂) with different aldehyde substrates and a potent aldehyde dehydrogenase inhibitor, disulfiram. TXB₂ was a competitive inhibitor of the aldehyde dehydrogenase reaction in assays with 3,4-dihydroxyphenylacetaldehyde, a high affinity substrate. The conversion of TXB₂ to 11-dehydro-TXB₂ was also inhibited by propanal and disulfiram.

The protein characteristics of the enzyme have also been further studied. The native enzyme is a tetramer and has an isoelectric point of 7.0 which is comparable with that of cytosolic aldehyde dehydrogenases from other species. Taken together the present data further indicate that 11-hydroxythromboxane B₂ dehydrogenase is identical with cytosolic aldehyde dehydrogenase and that substrates and inhibitors of aldehyde dehydrogenase interact with thromboxane metabolism in vitro.     

Thermodynamic regulation of human short-chain acyl-CoA dehydrogenase by substrate and product binding

Human short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle for substrates with four and six carbons. Previous studies have shown that the act of substrate/product binding induces a large enzyme potential shift in acyl-CoA dehydrogenases. The objective of this work was to examine the thermodynamic regulation of this process through direct characterization of the electrochemical properties of hSCAD using spectroelectrochemical methodology. A large amount of substrate activation was observed in the enzymatic reaction of hSCAD (+33 mV), the greatest magnitude measured in any acyl-CoA dehydrogenase to date. To examine the role of the substrate as well as the product in electron transfer by hSCAD, a catalytic base mutation (E368Q) was constructed. The E368Q mutation inactivates the reductive and oxidative pathways such that the individual effects of substrate and product binding on the redox potential can be investigated. Optimal substrate (butyryl-CoA) was seen to shift the flavin redox potential slightly more positive (+38 mV) than did optimal product (crotonyl-CoA) (+31 mV), a finding opposite of that observed in another short-chain enzyme, bacterial SCAD. These results indicate that substrate redox activation occurs in hSCAD leading to a large enzyme midpoint potential shift. Substrate binding in hSCAD appears to make a larger contribution than does product to thermodynamic modulation.     

Protonation of crotonyl-CoA dienolate by human glutaryl-CoA dehydrogenase occurs by solvent-derived protons

The protonation of crotonyl-CoA dienolate following decarboxylation of glutaconyl-CoA by glutaryl-CoA dehydrogenase was investigated. Although it is generally held that the active sites of acyl-CoA dehydrogenases are desolvated when substrate binds, recent evidence has established that water has access to the active site in these binary complexes of glutaryl-CoA dehydrogenase. The present investigation shows that the dehydrogenase catalyzes (a) a rapid exchange of C-4 methyl protons of crotonyl-CoA with bulk solvent and (b) protonation of crotonyl-CoA dienolate by solvent-derived protons under single turnover conditions. Both of the reactions require the catalytic base, Glu370. These findings indicate that decarboxylation proceeds via a dienolate intermediate. The involvement of water in catalysis by glutaryl-CoA dehydrogenase was previously unrecognized and is in conflict with a classically held intramolecular 1,3-prototropic shift for protonation of crotonyl-CoA dienolate.     

Intrinsic isomerase activity of medium-chain acyl-CoA dehydrogenase

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.     

Expression and purification of His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase wild-type and Arg256 mutant proteins

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. We cloned the gene of rat mitochondrial medium-chain acyl-CoA dehydrogenase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 3' of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 88% yield to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase was 4.0 U/mg. Arg256 is a highly conserved amino acid, which may play an important role in enzymatic reaction based on the crystal structure of medium-chain acyl-CoA dehydrogenase. We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Arg256 is a very important residue of rat mitochondrial medium-chain acyl-CoA dehydrogenase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial medium-chain acyl-CoA dehydrogenase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of medium-chain acyl-CoA dehydrogenase.     

Chemical mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

Homoisocitrate dehydrogenase (HIcDH, 3-carboxy-2-hydroxyadipate dehydrogenase) catalyzes the fourth reaction of the alpha-aminoadipate pathway for lysine biosynthesis, the conversion of homoisocitrate to alpha-ketoadipate using NAD as an oxidizing agent. A chemical mechanism for HIcDH is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. According to the pH-rate profiles, two enzyme groups act as acid-base catalysts in the reaction. A group with a p K a of approximately 6.5-7 acts as a general base accepting a proton as the beta-hydroxy acid is oxidized to the beta-keto acid, and this residue participates in all three of the chemical steps, acting to shuttle a proton between the C2 hydroxyl and itself. The second group acts as a general acid with a p K a of 9.5 and likely catalyzes the tautomerization step by donating a proton to the enol to give the final product. The general acid is observed in only the V pH-rate profile with homoisocitrate as a substrate, but not with isocitrate as a substrate, because the oxidative decarboxylation portion of the isocitrate reaction is limiting overall. With isocitrate as the substrate, the observed primary deuterium and (13)C isotope effects indicate that hydride transfer and decarboxylation steps contribute to rate limitation, and that the decarboxylation step is the more rate-limiting of the two. The multiple-substrate deuterium/ (13)C isotope effects suggest a stepwise mechanism with hydride transfer preceding decarboxylation. With homoisocitrate as the substrate, no primary deuterium isotope effect was observed, and a small (13)C kinetic isotope effect (1.0057) indicates that the decarboxylation step contributes only slightly to rate limitation. Thus, the chemical steps do not contribute significantly to rate limitation with the native substrate. On the basis of data from solvent deuterium kinetic isotope effects, viscosity effects, and multiple-solvent deuterium/ (13)C kinetic isotope effects, the proton transfer step(s) is slow and likely reflects a conformational change prior to catalysis.     

Inhibition of very long chain acyl-CoA dehydrogenase during cardiac ischemia

The heart utilizes primarily fatty acids for energy production. During ischemia, however, diminished oxygen supply necessitates a switch from beta-oxidation of fatty acids to glucose utilization and glycolysis. Molecular mechanisms responsible for these alterations in metabolism are not fully understood. Mitochondrial acyl-CoA dehydrogenase catalyzes the first committed step in the beta-oxidation of fatty acids. In the current study, an in vivo rat model of myocardial ischemia was utilized to determine whether specific acyl-CoA dehydrogenases exhibit ischemia-induced alterations in activity, identify mechanisms responsible for changes in enzyme function, and assess the effects on mitochondrial respiration. Very long chain acyl-CoA dehydrogenase (VLCAD) activity declined 34% during 30 min of ischemia. Loss in activity appeared specific to VLCAD as medium chain acyl-CoA dehydrogenase activity remained constant. Loss in VLCAD activity during ischemia was not due to loss in protein content. In addition, activity was restored in the presence of the detergent Triton X-100, suggesting that changes in the interaction between the protein and inner mitochondrial membrane are responsible for ischemia-induced loss in activity. Palmitoyl-carnitine supported ADP-dependent state 3 respiration declined as a result of ischemia. When octanoyl-carnitine was utilized state 3 respiration remained unchanged. State 4 respiration increased during ischemia, an increase that appears specific to fatty acid utilization. Thus, VLCAD represents a likely site for the modulation of substrate utilization during myocardial ischemia. However, the dramatic increase in mitochondrial state 4 respiration would be predicted to accentuate the imbalance between energy production and utilization.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.     

Structural studies on 10-hydroxygeraniol dehydrogenase: A novel linear substrate-specific dehydrogenase from Catharanthus roseus

Conversion of 10-hydroxygeraniol to 10-oxogeranial is a crucial step in iridoid biosynthesis. This reaction is catalyzed by a zinc-dependent alcohol dehydrogenase, 10-hydroxygeraniol dehydrogenase, belonging to the family of medium-chain dehydrogenase/reductase (MDR). Here, we report the crystal structures of a novel 10-hydroxygeraniol dehydrogenase from Catharanthus roseus in its apo and nicotinamide adenine dinucleotide phosphate (NADP⁺) bound forms. Structural analysis and docking studies reveal how subtle conformational differences of loops L1, L2, L3, and helix α9' at the orifice of the catalytic site confer differential activity of the enzyme toward various substrates, by modulating the binding pocket shape and volume. The present study, first of its kind, provides insights into the structural basis of substrate specificity of MDRs specific to linear substrates. Furthermore, comparison of apo and NADP⁺ bound structures suggests that the enzyme adopts open and closed states to facilitate cofactor binding.     

Structural basis for the substrate specificity of 3-hydroxybutyrate dehydrogenase

The substrate specificity of 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis with a non-native substrate, levulinic acid, was studied by analysis of the enzyme-substrate molecular interactions. The relation between structural and kinetic parameters was investigated considering the catalytic mechanism of the enzyme. The effects of key positive mutations (H144L, H144L/W187F) on the catalytic activity of the enzyme were studied by employing a surface analysis of its interatomic contacts between the enzyme and substrate atoms. The results revealed that the alteration of hydrogen bond network and rearrangement of the hydrophobic interactions between the active site and substrate molecule are the key structural basis for the change of the substrate specificity of 3-hydroxybutyrate dehydrogenase toward levulinic acid. With this approach, the structural basis for the substrate specificity of the enzyme could be elucidated in a quantitative manner.     

Substrate specificity analysis and inhibitor design of homoisocitrate dehydrogenase

Homoisocitrate dehydrogenase is involved in the alpha-aminoadipate pathway of biosynthesis of l-lysine in fungi, yeast, some prokaryotic bacteria, and archaea. This enzyme catalyzes the oxidative decarboxylation of (2R,3S)-homoisocitrate into 2-oxoadipate using NAD(+) as a coenzyme. Substrate specificity of two homoisocitrate dehydrogenases derived from Deinococcus radiodurans and Saccharomyces cerevisiae was analyzed using a series of synthetic substrate analogs, which indicated a relatively broad substrate specificity of these enzymes. Based on the substrate specificity, 3-hydroxyalkylidene- and 3-carboxyalkylidenemalate derivatives were designed as a specific inhibitor for homoisocitrate dehydrogenase. The synthetic inhibitors showed a moderate competitive inhibitory activity and (R,Z)-3-carboxypropylidenemalate was the most inhibitory among the synthesized inhibitors. Therefore, homoisocitrate dehydrogenase appeared to recognize preferentially an extended conformation of homoisocitrate.     

Identification and properties of an inducible phenylacyl-CoA dehydrogenase in Pseudomonas putida KT2440

A novel acyl-CoA dehydrogenase that initiates beta-oxidation of the side chains of phenylacyl-CoA compounds by Pseudomonas putida was induced by growth with phenylhexanoate as carbon source. It was identified as the product of gene PP_0368, which was cloned and overexpressed in Escherichia coli. This phenylacyl-CoA dehydrogenase was found to be dimeric with a subunit molecular mass of 66 kDa, to contain FAD and to be active with phenylacyl-CoA substrates having side chains from four to at least 11 carbon atoms. The same enzyme was induced by the aliphatic alkanoate octanoate. The optimal aliphatic substrates for the enzyme were palmitoyl-CoA and stearoyl-CoA, a property shared with mammalian very-long-chain acyl-CoA dehydrogenases. The FAD in the enzyme was reduced by aromatic and aliphatic substrates, with changes to the oxidation-reduction potential. Chemical reduction by dithionite ion and oxidation by ferricyanide ion showed that the enzyme can accept four electrons: two to reduce the flavin and two to slowly reduce an unknown acceptor, which in its reduced form interacts with the oxidized flavin in a charge-transfer complex. The experiments identify for the first time an acyl-CoA dehydrogenase that oxidizes the activated forms of aromatic acids similar to those used to first demonstrate the biological beta-oxidation of fatty acids.     

A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.     

The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization

The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.     

Mitochondrial aldehyde dehydrogenase from higher plants

Aldehyde dehydrogenase has been purified to homogeneity from mitochondria of potato tubers and pea epicotyls. Although the enzyme had a high affinity for glycolaldehyde it also had a high affinity for a number of other aliphatic and arylaldehydes. It is proposed that the codification glycolaldehyde dehydrogenase (EC 1.2.1.22) should be abandoned in favour of mitochondrial aldehyde dehydrogenase (EC 1.2.1.3). The purified enzyme showed esterase activity and had properties similar to those reported for the mammalian mitochondrial aldehyde dehydrogenase. Although the natural substrate(s) for the enzyme is not known, the kinetic properties of the enzyme are consistent with it playing a role in the oxidation of acetaldehyde, glycolaldehyde and indoleacetaldehyde.