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PubMed2004

Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid.

Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.

Bacterial Proteins↗
PubMed2006

Regulatory roles of the N-terminal domain based on crystal structures of human pyruvate dehydrogenase kinase 2 containing physiological and synthetic ligands.

Pyruvate dehydrogenase kinase (PDHK) regulates the activity of the pyruvate dehydrogenase multienzyme complex. PDHK inhibition provides a route for therapeutic intervention in diabetes and cardiovascular disorders. We report crystal structures of human PDHK isozyme 2 complexed with physiological and synthetic ligands. Several of the PDHK2 structures disclosed have C-terminal cross arms that span a large trough region between the N-terminal regulatory (R) domains of the PDHK2 dimers. The structures containing bound ATP and ADP demonstrate variation in the conformation of the active site lid, residues 316-321, which enclose the nucleotide beta and gamma phosphates at the active site in the C-terminal catalytic domain. We have identified three novel ligand binding sites located in the R domain of PDHK2. Dichloroacetate (DCA) binds at the pyruvate binding site in the center of the R domain, which together with ADP, induces significant changes at the active site. Nov3r and AZ12 inhibitors bind at the lipoamide binding site that is located at one end of the R domain. Pfz3 (an allosteric inhibitor) binds in an extended site at the other end of the R domain. We conclude that the N-terminal domain of PDHK has a key regulatory function and propose that the different inhibitor classes act by discrete mechanisms. The structures we describe provide insights that can be used for structure-based design of PDHK inhibitors.

Adenosine Triphosphate↗
PubMed1999

Nativelike structure and stability in a truncation mutant of a protein minidomain: the peripheral subunit-binding domain.

Despite its small size, the peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex adopts a unique, compact structure. To determine whether the full 43 residue sequence is required for the domain to adopt a stable, nativelike structure, 3 proteins of different lengths were prepared. Psbd41 corresponds to residues 3-43 of the domain, psbd36 spans residues 6-41, and psbd33 comprises residues 7-39. Psbd41 folds in a cooperative, two-state fashion with a Tm of 53 degrees C and a stability at 25 degrees C of 2.2 kcal mol-1. Psbd36 is nearly as stable with a Tm of 48 degrees C and a stability of 1.8 kcal mol-1. Similar m-values and heat capacities suggest that psbd36 and psbd41 bury approximately the same surface area. Minimal differences in CalphaH and NH chemical shifts between psbd41 and psbd36 show that the two sequences adopt the same tertiary fold. On a per residue basis, DeltaH degrees and DeltaC degrees p fall within the range typical for single-domain globular proteins. Psbd33 is significantly less stable. It is not fully folded at 25 degrees C, and at all temperatures it shows broadened NMR lines. ANS titrations provide evidence that this is due to an equilibrium between nativelike and unfolded molecules rather than formation of a molten globule. The fraction of psbd33 molecules which are folded appear to adopt the same structure as the full-length domain. Thus, although more than the 33 residue core is required to form a fully stable native structure, the entire sequence is not required for folding.

Amino Acid Sequence↗
PubMed2000

Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin?

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin.

Animals↗
PubMedDate not supplied

Comparative analysis of binding energy of chymostatin with human cathepsin A and its homologous proteins by molecular orbital calculation.

Cathepsin A is a mammalian lysosomal enzyme that catalyzes the hydrolysis of the carboxy-terminal amino acids of polypeptides and also regulates beta-galactosidase and neuraminidase-1 activities through the formation of a multienzymic complex in lysosomes. Human cathepsin A (hCathA), yeast carboxypeptidase (CPY), and wheat carboxypeptidase II (CPW) belong to the alpha/beta-hydrolase fold family. They have structurally similar active-site clefts, but there are small differences in the amino acid residues comprising their active sites that might determine the substrate specificity and sensitivity to microbial inhibitors including chymostatin. To examine the selectivity and binding mechanism of chymostatin as to hCathA, CPY, and CPW at the atomic level, we analyzed the interaction energy between chymostatin and each protein quantitatively by semiempirical molecular orbital calculation AM1 with the continuum solvent model. We predicted the electrostatic repulsion between the P3 cyclic arginine residue of the inhibitor and the Arg344 in the S3 active subsite of hCathA. Genetic conversion of Arg344 of the wild-type hCathA to Ile also caused an increase in its sensitivity to chymostatin, which was correlated with the decrease in the interaction energy calculated with the molecular orbital method. The present results suggest that such molecular calculation should be useful for evaluating the interactions between ligands, including inhibitors and homologous enzymes, in their docking models.

Base Sequence↗
PubMed2000

Metabolic functions of microbial nucleoside diphosphate kinases.

This article summarizes research from our laboratory on two aspects of the biochemistry of nucleoside diphosphate kinase from Escherichia coli--first, its interactions with several T4 bacteriophage-coded enzymes, as part of a multienzyme complex for deoxyribonucleoside triphosphate biosynthesis. We identify some of the specific interactions and discuss whether the complex is linked physically or functionally with the T4 DNA replication machinery, or replisome. Second, we discuss phenotypes of an E. coli mutant strain carrying a targeted deletion of ndk, the structural gene for nucleoside diphosphate kinase. How do bacteria lacking this essential housekeeping enzyme synthesize nucleoside triphosphates? In view of the specific interactions of nucleoside diphosphate kinase with T4 enzymes of DNA metabolism, how does T4 multiply after infection of this host? Finally, the ndk disruption strain has highly biased nucleoside triphosphate pools, including elevations of the CTP and dCTP pools of 7- and 23-fold, respectively. Accompanied by these biased nucleotide pools is a strong mutator phenotype. What is the biochemical basis for the pool abnormalities and what are the mutagenic mechanisms? We conclude with brief references to related work in other laboratories.

Bacteriophage T4↗
PubMed2001

Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana.

Plant fatty acid elongase which catalyzes very-long-chain fatty acid (VLCFA) biosynthesis is a membrane-bound multienzyme complex. It is composed of four enzymes, a 3-ketoacyl-CoA synthase (condensing enzyme), a 3-ketoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrase, and an enoyl-CoA reductase required for completion of each step of 2-carbon elongation of fatty acids. To improve our understanding of the overall regulation of the fatty acid elongase, we investigated the spatial and temporal expression of its key component, the FAE1-condensing enzyme, and examined the activity of the promoter of the FAE1 gene in Arabidopsis. In situ hybridization results revealed that FAE1 transcripts were found exclusively in the embryo. RNA blot analysis and histochemical analysis of GUS activity in pFAE1::GUS transgenic Arabidopsis lines demonstrated that the FAE1 gene was already transcribed in the early torpedo stage embryos 4-5 days after flowering, with transcription reaching its peak 9-11 days after flowering. VLCFA deposition closely paralleled FAE1 transcript accumulation. FAE1 promoter was highly active and embryo-specific. Because its timing coincides with the period of major storage lipid accumulation, and because its in vivo activity in Arabidopsis is superior to the napin promoter, FAE1 promoter may be ideal for genetic engineering of seed oil composition.

Acetyltransferases↗
PubMed2002

Understanding the stepwise synthesis of glycolipids.

Glycolipid expression is highly regulated during development and differeniation. The control relies mainly on transcriptional modulation of key glycosyltransferases acting at the branching points of the pathway of biosynthesis. Transferases are Golgi residents that depend on N-glycosylation and oligosaccharide processing for proper folding in the endoplasmic reticulum. The N-terminal domain bears information for their transport to the Golgi, retention in the organelle and differential concentration in sub-Golgi compartments. In the Golgi, some transferases associate forming functional multienzyme complexes. It is envisaged that the machinery for synthesis in the Golgi complex, and its dynamics, constitute a potential target for fine tuning of the control of glycolipid expression according to cell demands.

Animals↗
PubMed2003

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1alpha subunit.

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B1 therapy. T231A has very low activity and a greatly elevated Km for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

Acidosis, Lactic↗
PubMedDate not supplied

On the origin of intracellular compartmentation and organized metabolic systems.

The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly determines the major characteristics of cellular environment such as viscosity, diffusion and inhomogeneity. The fact that the solvent viscosity of cytoplasm is not substantially different from the water is explained by intracellular structural heterogeneity: the intrinsic macromolecular density is relatively low within the interstitial voids in the cell because many soluble enzymes are apparently integral parts of the insoluble cytomatrix and are not distributed homogeneously. The molecular crowding and sieving restrict the mobility of very large solutes, binding severely restrict the mobility of smaller solutes. One of consequence of molecular crowding and hindered diffusion is the need to compartmentalize metabolic pathway to overcome diffusive barriers. Although the movement of small molecules is slowed down in the cytoplasm, the metabolism can successfully proceed and even be facilitated by metabolite channeling which directly transfers the intermediate from one enzyme to an adjacent enzyme without the need of free aqueous-phase diffusion. The enhanced probability for intermediates to be transferred from one active site to the other by sequential enzymes requires stable or transient interactions of the relevant enzymes, which associate physically in non-dissociable, static multienzyme complexes--metabolones, particles containing enzymes of a part or whole metabolic systems. Therefore, within the living cell the metabolism depends on the structural organization of enzymes forming microcompartments. Since cells contain many compartments and microenvironments, the measurement of the concentration of metabolites in whole cells or tissues gives an average cellular concentration and not that which is actually sensed by the active site of a specific enzyme. Thus, the microcompartmentation could provide a mechanism which can control metabolic pathways. Independently and in parallel to the developments described above, the ideas of compartmentation came into existence from the necessity to explain important physiological phenomena, in particular in heart research and in cardiac electrophysiology. These phenomena demonstrated the physiological importance of the biophysical and biochemical mechanisms described in this review.

Cell Compartmentation↗
PubMed2000

The peculiar autoimmunity of primary biliary cirrhosis.

Autoantibodies to mitochondria (AMA, anti-M2) are a serologic hallmark of primary biliary cirrhosis (PBC). These react with three structurally and functionally related multienzymic complexes, the 2-oxoacid dehydrogenase complexes, but chiefly with the E2 subunit of pyruvate dehydrogenase complex (PDC-E2). Their very dose (95%) and specific association with PBC underpins the autoimmune concept of pathogenesis of that disease, notwithstanding several non-congruent features. Detailed studies, including structural analysis of epitopes, do not disclose how these autoantibodies originate. Their ubiquity in PBC has overshadowed the existence of a second set of relatively PBC-specific autoantibodies to nuclear antigens for which reactants have been cloned and characterized. These include centromeric proteins; proteins of the nuclear pore complex; nuclear dot proteins, which include Sp-100 and the promyelocytic leukemia antigen; and a recently identified autoantigen, SOX13. Certain of these reactants are DNA-binding proteins with transcriptional regulatory activity. Thus serum from individuals with the same clinical syndrome can have autoimmune reactivity to disparate mitochondrial and nuclear constituents in different cellular compartments. Antibody probing of phage displayed random peptide libraries, together with epitope scanning using overlapping sequential octameric peptides from the PDC-E2 sequence, showed that the discontinuous motifs MH, FV(E) and SYP contributed to a predicted conformational antibody epitope in the inner lipoyl domain of PDC-E2.

Acyltransferases↗
PubMed2006

Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels.

Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.

Binding Sites↗
PubMed2003

Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions.

The mechanism of thiamin diphosphate-dependent enzymatic reactions is discussed, concentrating on two enzymes involved in decarboxylating pyruvic acid, the yeast pyruvate decarboxylase and the pyruvate dehydrogenase multienzyme complex from Escherichia coli. The availability of high-resolution X-ray structures for several thiamin diphosphate-dependent enzymes, the use of site-specifically substituted protein variants (resulting from site-directed mutagenesis), the development of model reactions for the various putative intermediates, and the application of new mechanistic tools in solution have all contributed to a much better understanding of the role of the protein component in catalysis. Perhaps the most important advance in our understanding of these mechanisms concerns the role of the 4'-aminopyrimidine component of the coenzyme, widely ignored prior to the publication of the X-ray results. The current view is that the two aromatic rings both contribute to catalysis, perhaps carrying out an intramolecular proton transfer to initiate the various reactions, an ability that makes this coenzyme virtually unique among coenzymes.

Catalysis↗
PubMed2004

Determination of the binding specificity of the 12S subunit of the transcarboxylase by saturation transfer difference NMR.

In this study we present the characterization of the interaction of biotin and methylmalonyl-CoA (MMCoA) with the carboxyltransferase subunit (12S) from the transcarboxylase (TC) from Propionibacterium shermanii. This biotin dependent multienzyme complex catalyses the transfer of carbon dioxide from methylmalonyl-CoA (MMCoA) to pyruvate. The Saturation Transfer Difference NMR (STD) technique was performed to determine the binding epitope from biotin and MMCoA to the 12S subunit. We could show by titrations during STD experiments that biotin and MMCoA bind cooperatively in one binding pocket.

Acyl Coenzyme A↗
PubMed1972

Magnesium protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Studies with whole cells.

1. Whole cells of Rhodopseudomonas spheroides grown under semi-anaerobic conditions in the light incorporated magnesium into exogenous protoporphyrin when incubated with EDTA or the related chelators EGTA, N-(2-hydroxyethyl)-ethylenediamine-NN'N'- triacetate and trans-1,2-diaminocyclohexanetetra-acetate. 2. The reaction was demonstrated under anaerobic conditions in the light or at low oxygen partial pressure in the dark. Partial pressures of oxygen greater than 15% inhibited the reaction. 3. Cells grown under pure oxygen were completely inactive, but on adaptation to growth under low oxygen partial pressure (O(2)+N(2), 5:95) the development of activity paralleled the synthesis of bacteriochlorophyll. 4. The reaction with normal cells did not require protein synthesis, but cells that had lost their activity by being illuminated in Mg(2+)-deficient medium did not recover it in the absence of protein synthesis. 5. The product of the reaction was magnesium protoporphyrin monomethyl ester. 6. Evidence is presented that insertion of magnesium is obligatorily coupled with methylation and it is concluded that the reaction is dependent on a multienzyme complex.

Acetates↗
PubMed1973

Intermediates in fatty acid oxidation.

1. Aqueous extracts of acetone-dried liver and kidney mitochondria, supplemented with NAD(+), CoA and phenazine methosulphate, efficiently convert fatty-acyl-CoA compounds into acetyl-CoA; the process was followed with an O(2) electrode. 2. Label from [1-(14)C]octanoyl-CoA appears in acetyl-CoA more rapidly than that from [8-(14)C]octanoyl-CoA. 3. Oxidation of [8-(14)C]octanoyl-CoA was terminated by addition of neutral ethanolic hydroxylamine and the resulting hydroxamates were separated chromatographically. Hydroxamate derivatives of 3-hydroxyoctanoyl-, hexanoyl-, butyryl- and acetyl-CoA were obtained. 4. These and other observations suggest that oxidation of octanoyl-CoA by extracts involves participation of free intermediates rather than uninterrupted complete degradation of individual molecules to acetyl-CoA by a multienzyme complex. 5. Intact liver mitochondria studied by the hydroxamate technique were also shown to form intermediates during oxidation of labelled octanoates. In addition to octanoylhydroxamate, [8-(14)C]octanoate gave rise to small amounts of hexanoyl-, butyryl- and 3-hydroxyoctanoyl-hydroxamate. In contrast with extracts, however, where the quantity of intermediates found was a significant fraction of the precursors, mitochondria oxidizing octanoate contained much larger quantities of octanoyl-CoA than of any other intermediate.

Acetyl Coenzyme A↗
PubMed1980

Isotope-dilution analysis of the effects of deoxyguanosine and deoxyadenosine on the incorporation of thymidine and deoxycytidine by hydroxyurea-treated thymus cells.

It is presumed that the dGTP and dATP needed for replicative DNA synthesis can be formed by way of either ;salvage' pathways or biosynthesis de novo. This was examined by adding hydroxyurea to cultures of rat thymus cells to inhibit ribonucleoside diphosphate reductase, a key enzyme of the ;de novo' pathway. Most of the inhibition of the incorporation of [Me-(3)H]thymidine and deoxy[5-(3)H]cytidine by low concentrations of hydroxyurea (100-500mum) was prevented by substrates of the salvage pathway (400mum-deoxyguanosine and, to a lesser extent, 200mum-deoxyadenosine). However, isotope-dilution studies indicated that the purine deoxyribonucleosides prevented inhibition by decreasing pyrimidine deoxyribonucleotide competitor pools. Evidence was obtained that a hydroxyurea-induced increase in the thymidine-competitor pool (probably dTTP) was prevented to an equal extent by deoxyguanosine and by the inhibitor of thymidylate synthase, deoxy-5-fluorouridine. These compounds had almost identical effects on hydroxyurea dose-response curves and on thymidine isotope-dilution plots. The evidence suggests that exogenous purine deoxyribonucleosides cannot prevent the inhibition by hydroxyurea of thymus-cell DNA synthesis. This could mean that, with respect to the metabolism of purine deoxyribonucleotides, ribonucleoside diphosphate reductase is tightly coupled to DNA polymerase in a multienzyme complex. The complex would not permit entry of exogenous metabolic intermediates into the ;de novo' pathway, but would still be subject to the regulatory effects of these intermediates. Thus dGTP and dATP formed from exogenous purine deoxyribonucleosides by salvage pathways might deplete pyrimidine deoxyribonucleotide competitor pools by inhibiting relatively hydroxyurea-insensitive activities of ribonucleoside diphosphate reductase.

Animals↗