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PubMed1988

Nucleotide sequence of a cDNA for branched chain acyltransferase with analysis of the deduced protein structure.

Nucleotide sequence was determined for a 1.6-kilobase human cDNA putative for the branched chain acyltransferase protein of the branched chain alpha-ketoacid dehydrogenase complex. Translation of the sequence reveals an open reading frame encoding a 315-amino acid protein of molecular weight 35,759 followed by 560 bases of 3'-untranslated sequence. Three repeats of the polyadenylation signal hexamer ATTAAA are present prior to the polyadenylate tail. Within the open reading frame is a 10-amino acid fragment which matches exactly the amino acid sequence around the lipoate-lysine residue in bovine kidney branched chain acyltransferase, thus confirming the identity of the cDNA. Analysis of the deduced protein structure for the human branched chain acyltransferase revealed an organization into domains similar to that reported for the acyltransferase proteins of the pyruvate and alpha-ketoglutarate dehydrogenase complexes. This similarity in organization suggests that a more detailed analysis of the proteins will be required to explain the individual substrate and multienzyme complex specificity shown by these acyltransferases.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1994

Coordination of catalytic activities within enzyme complexes.

If two enzymes are physically and permanently associated as a bi-enzyme complex and if these enzymes catalyze non-consecutive chemical reactions, either of these reactions may inhibit or activate the other. If these reactions belong to two different metabolic cycles, the functioning of one of these cycles will control the fine tuning of the other. Thus simple kinetic considerations lead to the conclusion that, owing to the spatial organization of enzymes as multimolecular complexes, a fine tuning and a coordination of different metabolic networks, or cycles, may be exerted. It thus appears that channelling of reaction intermediates within a multienzyme complex does not represent the only functional advantage brought about by this type of spatial molecular organization.

Catalysis↗
PubMed2002

Histidine 407, a phantom residue in the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex, activates reductive acetylation of lipoamide on the E2 subunit. An explanation for conservation of active sites between the E1 subunit and transketolase.

Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductive acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed lipoyl domain and of the intact 1-lipoyl E2 by the H407A variant compared to the parental E1. A proposal is presented to explain the remarkable conservation of the three-dimensional structure at the active centers of the E. coli E1 subunit and transketolase on the basis of the parallels in the ligation-type reactions carried out and the need to protonate a very weak acid, a dithiolane sulfur atom in the former, and a carbonyl oxygen atom in the latter.

Acetylation↗
PubMed1998

Growth medium-dependent regulation of Myxococcus xanthus fatty acid content is controlled by the esg locus.

We compared the cellular fatty acid profiles of Myxococcus xanthus cells grown in either a Casitone-based complex medium or a chemically defined medium. The cells grown in the complex medium had a much higher content of the abundant branched-chain fatty acid iso-15:0 and several other branched-chain species. The higher branched-chain fatty acid content of the cells grown in the complex medium was dependent on the esg locus, which encodes the E1alpha and E1beta components of a branched-chain keto acid dehydrogenase (BCKAD) multienzyme complex involved in branched-chain fatty acid biosynthesis. Cells grown in the complex medium were also found to have a higher level of esg transcription and more BCKAD enzyme activity than cells from the chemically defined medium. The level of esg transcription appears to be an important factor in the growth medium-dependent regulation of the M. xanthus branched-chain fatty acid content.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1980

Studies on the activation and inactivation of the branched chain alpha-keto acid dehydrogenase in the perfused rat heart.

Evidence for a reversible process resulting in stable activated and inactivated states of the mitochondrial branched chain alpha-keto acid dehydrogenase complex in isolated perfused rat heart is presented. The inactivation process is mediated by pyruvate infusion, while activation (up to 18-fold) is facilitated by branched chain alpha-keto acid substrates. The low activity state of the branched chain complex characteristic of freshly excised rat hearts could be maintained by infusion of either pyruvate or glucose. Activation of the complex in the perfused rat heart was achieved slowly by substrate-free perfusion, while rapid activation was accomplished by infusion of branched chain alpha-keto acids. The fully activated enzyme complex resulting from branched chain alpha-keto acid infusion subsequently could be inactivated maximally by infusion of pyruvate alone or intermediate degrees of inactivation could be produced by certain ratios of co-infused pyruvate and branched chain alpha-keto acid. alpha-Ketoisocaproate was an order of magnitude more effective than alpha-keto isovalerate either in preventing inactivation or in stimulating the opposing activation process when co-infused with pyruvate. The mitochondrial pyruvate transport inhibitor, alpha-cyanocinnamate, effectively prevented inactivation of the complex by infused pyruvate. Differential changes in the activation states of the branched chain alpha-keto acid dehydrogenase and pyruvate dehydrogenase complexes were evident when the two complexes were compared in apparently similar flux-inhibited (via octanoate infusion) and flux-stimulated (via dichloroacetate infusion) metabolic conditions. The differential effect of pyruvate concentration on the activity states of the two complexes was also well-defined. The results of the present study suggest distinct systems for the regulation of the activity of the two multienzyme complexes of interest. While our results argue neither for nor against an inactivation of the branched chain alpha-keto acid dehydrogenase complex by a protein kinase, the regulatory properties of such an intramitochondrial protein kinase may not be similar to the pyruvate dehydrogenase kinase. The mechanistic nature of the suggested novel regulatory system concerned with the pyruvate-mediated inactivation of the branched chain alpha-keto acid activation cannot be inferred at the present time.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1982

Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. I. Species specificity of the polypeptide composition.

Using a three-step procedure designed to minimize the risks of proteolysis, high molecular weight complexes containing the same seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, lysine, arginine, glutamic acid, and glutamine were purified from sheep liver and spleen, as well as from rabbit reticulocytes and liver. The polypeptide composition of these complexes, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is characteristic of the animal species from which they are derived. The complexes from sheep liver and spleen display indistinguishable polypeptide patterns composed of 11 major components. Of the 10 common components which characterize the complexes of rabbit reticulocytes and liver, 4 are also shared by the complexes from sheep, while 6 have distinctly different electrophoretic mobilities. Furthermore, in the case of the complex from rabbit reticulocytes, it is shown that the enzyme and polypeptide composition of the complex is independent of the purification method employed. The isolation of high molecular weight complexes of identical aminoacyl-tRNA synthetase and polypeptide compositions from two cell types as radically different as rabbit reticulocytes and hepatocytes suggests that these multienzyme complexes do not arise as artifacts of preparation and supports the view that they reflect a structural organization existing within the cell.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Cloning and analysis of a cDNA encoding mammalian arginyl-tRNA synthetase, a component of the multisynthetase complex with a hydrophobic N-terminal extension.

In mammalian cells, the nine aminoacyl-tRNA synthetases (aaRS) specific for the amino acids (aa) Glu, Pro, Ile, Leu, Met, Gln, Lys, Arg and Asp are associated within a multienzyme complex. Arginyl-tRNA synthetase (ArgRS) is characterized by the occurrence of two structurally distinct forms of that enzyme: a complexed (approximately 74 kDa) and a free (approximately 60 kDa) form. The cDNA encoding the 74-kDa species of ArgRS from Chinese hamster ovary cells has been isolated and sequenced. The deduced aa sequence shows 38% identity to the homologous bacterial enzyme but displays an N-terminal polypeptide extension composed of 73 aa, which is absent in the free form of mammalian ArgRS. Two regions of this extension are predicted to be alpha-helical, leading to the clustering of Leu and Ile residues on one side of the helices. This suggests that the N-terminal domain is involved in the assembly of the 74-kDa species of ArgRS within the multisynthetase complex through hydrophobic interactions. By using the isolated cDNA, a Northern blot analysis showed a single mRNA species. Thus, there is a possibility that the free and complexed forms of ArgRS are encoded by the same gene.

Amino Acid Sequence↗
PubMed1989

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex.

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.

Animals↗
PubMed1997

A polyketide-synthase-like gene is involved in the synthesis of heterocyst glycolipids in Nostoc punctiforme strain ATCC 29133.

A Tn5-1063-derived mutant of Nostoc punctiforme strain ATCC 29133 was unable to fix N2 in air although it reduced acetylene in the absence of O2. Mutant strain UCD 307 formed cells morphologically similar to heterocysts, but it failed to synthesize the characteristic heterocyst glycolipids. Sequence analysis around the site of insertion revealed an ORF of 3,159 base pairs, termed hglE. hglE putatively encodes a 115.4-kDa protein containing two domains with conserved amino acid sequences identified with acyl transferase and the chain length factor variation of beta-ketoacyl synthase active sites. These active sites are characteristic of polyketide and fatty acid synthases. The N. punctiforme strain 29133 hglE gene is transcribed only under nitrogen-limiting growth conditions. The hglE gene, or similar sequences, was found in all other heterocyst-forming cyanobacteria surveyed and was absent in unicellular Synechococcus sp. strain PCC 7942. Based on these results, we propose that the synthesis of heterocyst glycolipids follows a pathway characteristic of polyketide synthesis and involves similar large, multienzyme complexes.

Amino Acid Sequence↗
PubMed1992

Multienzyme membranes for biosensors.

Artificial multienzyme complexes were prepared in which enzymes were covalently bound to polysaccharide structures activated with urea and formaldehyde. Double enzyme complexes of glucose oxidase and catalase, a glucose oxidase and invertase, were prepared by immobilization on to cellulose fabric. Also, catalase was covalently bound to soluble dextran. The resulting multienzyme systems were highly active and stable, making them suitable for use in measuring the concentrations of glucose and saccharose in solutions. The measurements were performed using an amperometric oxygen electrode and multienzyme membranes containing glucose oxidase and catalase for the first substrate, as well as glucose oxidase bound to cheese-cloth and a 'liquid' membrane of dextran-bound catalase. To determine the concentration of saccharose, a multienzyme membrane with bound glucose oxidase and invertase was used in combination with a 'liquid' dextran-catalase. The enzyme electrodes exhibited a measuring range of 0.1-5 mol dm-3 and a response time of 2-3 min. The electrodes may be used for measuring saccharose and glucose concentrations both in fermentation broths and food products.

Biosensing Techniques↗
PubMed1995

Structure and expression analysis of the gdcsPA and gdcsPB genes encoding two P-isoproteins of the glycine-cleavage system from Flaveria pringlei.

In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.

Amino Acid Oxidoreductases↗
PubMed1993

Substrate and cofactor reactivity of a carbon monoxide dehydrogenase-corrinoid enzyme complex: stepwise reduction of iron-sulfur and corrinoid centers, the corrinoid Co2+/1+ redox midpoint potential, and overall synthesis of acetyl-CoA.

Cleavage of the acetyl carbon-carbon bond of acetyl-CoA in Methanosarcina barkeri is catalyzed by a high molecular mass multienzyme complex. The complex contains a corrinoid protein and carbon monoxide dehydrogenase and requires tetrahydrosarcinapterin (H4SPt) as methyl group acceptor. Reactions of the enzyme complex with carbon monoxide and with the methyl group donor N5-methyltetrahydrosarcinapterin (CH3-H4SPt) have been analyzed by UV-visible spectroscopy. Reduction of the enzyme complex by CO occurred in two steps. In the first step, difference spectra exhibited peaks of maximal absorbance decrease at 426 nm (major) and 324 nm (minor), characteristic of Fe-S cluster reduction. In the second step, corrinoid reduction to the Co1+ level was indicated by a prominent peak of increased absorbance at 394 nm. Spectrophotometric analyses of the corrinoid redox state were performed on the intact complex at potentials poised by equilibration with gas mixtures containing different [CO2]/[CO] ratios or by variation of the [H+]/[H2] ratio. The corrinoid Co2+/1+ midpoint potential was -426 mV (+/- 4 mV, n = 1.16 electrons, 24 degrees C), independent of pH (pH 6.4-8.0). The results indicated a significant fraction of Co1+ corrinoid at potentials existing in vivo. The reduced corrinoid reacted very rapidly with CH3-H4SPt. Reaction with methyl iodide was slow, and methylation by S-adenosylmethionine was not observed. Tne rate of methyl group transfer from CH3-H4SPt greatly exceeded the rate of CO reduction of enzyme centers. The enzyme complex catalyzed efficient synthesis of acetyl-CoA from coenzyme A, CO, and CH3-H4SPt. During acetyl-CoA synthesis, demethylation of CH3-H4SPt was monitored by the absorbance increase at 312 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl Coenzyme A↗
PubMed1994

Specific interaction of penicillin-binding proteins 3 and 7/8 with soluble lytic transglycosylase in Escherichia coli.

Soluble lytic transglycosylase 70 (Slt70), one of the better characterized murein hydrolases of Escherichia coli, was covalently bound to CNBr-activated Sepharose and used as a specific tool to screen for proteins showing an affinity for Slt70. Several proteins were specifically enriched by Slt-Sepharose affinity chromatography. Two of them were identified as the penicillin-binding proteins (PBP)3 and PBP7/8. Thus, the bifunctional synthase PBP3, specifically involved in septum formation, and PBP7/8, recently shown to be a DD-endopeptidase, bind to Slt70 in vitro. In addition, PBP7/8 was found not only to stabilize but also to stimulate the enzymatic activity of Slt70 by a protein-protein interaction. It is concluded that Slt70, PBP7/8, and PBP3 may form a multienzyme complex in vivo.

Bacterial Proteins↗
PubMed2002

The complex fate of alpha-ketoacids.

Plant cells are unique in that they contain four species of alpha-ketoacid dehydrogenase complex: plastidial pyruvate dehydrogenase, mitochondrial pyruvate dehydrogenase, alpha-ketoglutarate (2-oxoglutarate) dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. All complexes include multiple copies of three components: an alpha-ketoacid dehydrogenase/decarboxylase, a dihydrolipoyl acyltransferase, and a dihydrolipoyl dehydrogenase. The mitochondrial pyruvate dehydrogenase complex additionally includes intrinsic regulatory protein-kinase and -phosphatase enzymes. The acyltransferases form the intricate geometric core structures of the complexes. Substrate channeling plus active-site coupling combine to greatly enhance the catalytic efficiency of these complexes. These alpha-ketoacid dehydrogenase complexes occupy key positions in intermediary metabolism, and a basic understanding of their properties is critical to genetic and metabolic engineering. The current status of knowledge of the biochemical, regulatory, structural, genomic, and evolutionary aspects of these fascinating multienzyme complexes are reviewed.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2001

The Na(+)-translocating methyltransferase complex from methanogenic archaea.

Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N(5)-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (DeltaG degrees '=-30 kJ/mol) is catalyzed by a Na(+)-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA-H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions.

Amino Acid Sequence↗
PubMed1999

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein.

Cathepsin A/protective protein [3.4.16.5], carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y. Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases. Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities. Cathepsin A was recently identified in human platelets as deamidase. In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions. Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function. The protective function of cathepsin A is distinct from its catalytic function. Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein'. Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase. Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A. The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis. Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A. Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.

Age of Onset↗
PubMed1981

Stoichiometry and composition of an aminoacyl-tRNA synthetase complex from rat liver.

The particulate aminoacyl-tRNA synthetases of rat liver were copurified about 1000-fold with more than 20% yields for individual synthetase activities. Measurements of aminoacylation activities showed that lysyl-, arginyl-, leucyl-, isoleucyl-, and methionyl-tRNA synthetases in the purified complex cosedimented at 18 S. The molecular weight of the synthetase complex is about one million, as estimated by gel filtration. The stoichiometry of the synthetase in the complex was determined by active site titration with aminoacyl adenylates. Results indicate that the 18S synthetase complex contains one subunit of methionyl-tRNA synthetase and two subunits of lysyl-tRNA synthetase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the 18S synthetase complex contains eight major protein bands. Proteins with subunit molecular weights of 104,000, 92,000, 69,000, and 67,000 are present in molar ratios of 1:1:2:2, while proteins with subunit molecular weights of amounts. These results suggest that the particulate aminoacyl-tRNA synthetases exist as a heterotypic multienzyme complex with defined structure.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Antibodies to bovine liver branched-chain 2-oxo acid dehydrogenase cross-react with this enzyme complex from other tissues and species.

Antibodies were produced in rabbits against bovine liver branched-chain 2-oxo acid dehydrogenase (BCOAD) for three reasons. First, since activity of BCOAD varies among tissues within a species, it is necessary to ascertain whether isoenzymes exist within the different tissues. Second, it is of great interest to determine the structural similarity of BCOAD among mammalian species. Third, heritable defects of this multienzyme complex are known in humans, and techniques for studying BCOAD subunit composition in cells expressing the defect include the use of specific antibodies. Antibodies produced against BCOAD inhibit complex activity and cross-react with BCOAD from various tissues and species. By using rocket immunoelectrophoresis, the complex can be quantified in mitochondrial extracts. Each subunit is immunogenic, making the detection of subunit variants possible by the combined techniques of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and Western blotting. Finally, antibodies produced against pig heart lipoamide dehydrogenase cross-react with BCOAD, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, giving further evidence for the commonality of this subunit among these three mitochondrial complexes.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗