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PubMed2003

Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core.

Transcarboxylase from Propionibacterium shermanii is a 1.2 MDa multienzyme complex that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate, yielding propionyl-CoA and oxaloacetate. The 1.9 A resolution crystal structure of the central 12S hexameric core, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl-CoA. Overall, the structure reveals two stacked trimers related by 2-fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl-CoA is stabilized by interaction with the N-termini of two alpha-helices. The 12S domains are structurally similar to the crotonase/isomerase superfamily, although only domain 1 of each 12S monomer binds ligand. The 12S reaction is similar to that of human propionyl-CoA carboxylase, whose beta-subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase beta-subunit, based on this 12S crystal structure, provides new insight into the propionyl-CoA carboxylase mechanism, its oligomeric structure and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.

Acyl Coenzyme A↗
PubMed1999

In vitro transcriptional studies of the bkd operon of Pseudomonas putida: L-branched-chain amino acids and D-leucine are the inducers.

BkdR is the transcriptional activator of the bkd operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas putida. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5' region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative rho-independent terminator of the bkd operon. Substrate DNA, BkdR, and any of the L-branched-chain amino acids or D-leucine was required for transcription. Branched-chain keto acids, D-valine, and D-isoleucine did not promote transcription. Therefore, the L-branched-chain amino acids and D-leucine are the inducers of the bkd operon. The concentration of L-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the bkd operon by BkdR during enzyme induction which incorporates these results is presented.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation.

Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.

Arabidopsis↗
PubMed2003

Regulation of the cellulosomal CelS (cel48A) gene of Clostridium thermocellum is growth rate dependent.

Clostridium thermocellum produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose. One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase. The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses indicated that the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures. The transcriptional level of celS mRNA was determined quantitatively by RNase protection assays with batch and continuous cultures under carbon and nitrogen limitation. The amount of celS mRNA transcripts per cell was about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 h(-1) and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 h(-1), respectively. Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 h(-1), respectively. Two major transcriptional start sites were detected at positions -140 and -145 bp, upstream of the translational start site of the celS gene. The potential promoters exhibited homology to known sigma factors (i.e., sigma(A) and sigma(B)) of Bacillus subtilis. The relative activity of the two promoters remained constant under the conditions studied and was in agreement with the results of the RNase protection assay, in which the observed transcriptional activity was inversely proportional to the growth rate.

Bacterial Proteins↗
PubMed1990

EntG activity of Escherichia coli enterobactin synthetase.

The last steps in the biosynthesis of the Escherichia coli siderophore enterobactin (Ent) are carried out by Ent synthetase, a multienzyme complex believed to be composed of the entD, -E, -F, and -G products (EntD to -G). However, sequencing data showed that there is no separate entG gene and, unlike EntD to -F, no distinct EntG polypeptide has been identified. In this study, genetic, biochemical, and immunological approaches were used to study the anomalies associated with EntG activity. Two plasmids, pJS43 and pJS100, were isolated that had mutations resulting in truncated EntB proteins; both had the phenotype EntB+ EntG-. PJS43 had a Tn5 inserted 198 bp from the entB termination codon, and pJS100 had the last 25 codons of entB deleted. Plasmids isolated with Tn5 insertions in the 5' half of entB had the phenotype EntB- EntG+. These latter Tn5 mutations were EntB- EntG- when moved to the bacterial chromosome. Polyclonal antiserum was prepared and shown to react only with intact EntB in Western immunoblots. Addition of anti-EntB antiserum to Ent synthetase assays resulted in complete inhibition of enzyme activity, whereas preimmune serum had no effect. Lastly, AN462, the type strain for entG which was derived by Mu insertion and which has the phenotype EntB-G-A-, was characterized. Southern blot data showed a Mu insertion, presumably with polar effects, in the vicinity of the 5' end of entB. In summary, EntG activity was found to be encoded by the entB 3' terminus. The evidence, while not rigorously eliminating the possibility that a separate EntG polypeptide exists, strongly supports the idea that EntB is a bifunctional protein.

Blotting, Southern↗
PubMed1993

Tracing intracellular proteolytic pathways. Proteolysis of fatty acid synthase and other cytoplasmic proteins in the yeast Saccharomyces cerevisiae.

Yeast fatty acid synthase consists of two independent polypeptide strains, alpha and beta. The functional multienzyme complex, composed of six alpha- and six beta-subunits, is rather stable against proteolysis in vivo. Mutations in one of the subunits or deletion of one subunit lead to degradation of the nonmutated remaining fatty acid synthase protein. We show that the unassembled alpha-subunit of this enzyme is short-lived, and degradation depends on the presence of active cytoplasmic proteinase yscE, the yeast proteasome. The unassembled beta-subunit is degraded by a nonvacuolar proteolytic system under vegetative growth conditions. However, starvation of a vacuolar proteinase mutant strain, which lacks the alpha-subunit of fatty acid synthase, leads to appearance of the unassembled beta-subunit is isolated vacuoles. This indicates that the major vacuolar peptidases proteinase yscA and yscB are at least partly involved in degradation of the beta-subunit of fatty acid synthase. In a proteinase yscA and yscB double mutant strain wild type for fatty acid synthase both subunits of fatty acid synthase, alpha and beta, are detectable in vacuoles. In addition, under the same starvation conditions other cytoplasmic proteins are found in the vacuole of a proteinase yscA and yscB double mutant strain. The experiments in conjunction with the previous finding of the appearance of vesicles in vacuoles of starved cells (Simeon, A., van der Klei, I.J., Veenhuis, M., and Wolf, D. H. (1992) FEBS Lett. 301, 231-235) indicate that transport of these tested cytoplasmic proteins into the vacuole is an unselective bulk process induced by nutritional stress.

Cysteine Endopeptidases↗
PubMed1999

Mathematical modeling and optimization of cellulase protein production using Trichoderma reesei RL-P37.

The enzyme cellulase, a multienzyme complex made up of several proteins, catalyzes the conversion of cellulose to glucose in an enzymatic hydrolysis-based biomass-to-ethanol process. Production of cellulase enzyme proteins in large quantities using the fungus Trichoderma reesei requires understanding the dynamics of growth and enzyme production. The method of neural network parameter function modeling, which combines the approximation capabilities of neural networks with fundamental process knowledge, is utilized to develop a mathematical model of this dynamic system. In addition, kinetic models are also developed. Laboratory data from bench-scale fermentations involving growth and protein production by T. reesei on lactose and xylose are used to estimate the parameters in these models. The relative performances of the various models and the results of optimizing these models on two different performance measures are presented. An approximately 33% lower root-mean-squared error (RMSE) in protein predictions and about 40% lower total RMSE is obtained with the neural network-based model as opposed to kinetic models. Using the neural network-based model, the RMSE in predicting optimal conditions for two performance indices, is about 67% and 40% lower, respectively, when compared with the kinetic models. Thus, both model predictions and optimization results from the neural network-based model are found to be closer to the experimental data than the kinetic models developed in this work. It is shown that the neural network parameter function modeling method can be useful as a "macromodeling" technique to rapidly develop dynamic models of a process.

Cellulase↗
PubMed1998

Catalytic self-acylation of type II polyketide synthase acyl carrier proteins.

BACKGROUND: Aromatic polyketides are synthesised in streptomycetes by the successive condensation of simple carboxylic acids, catalysed by multienzyme complexes--the polyketide synthases (PKSs). Polyketide assembly intermediates are covalently linked as thioesters to the holo-acyl carrier protein (ACP) subunit of these type II PKSs. The ACP is primed for chain elongation by the transfer of malonate from malonyl CoA. Malonylation of fatty acid synthase (FAS) ACPs is catalysed by specific malonyl transferase (MT) enzymes. The type II PKS gene clusters apparently lack genes encoding such MT proteins, however. It has been proposed that the MT subunit of the FAS in streptomycetes catalyses malonylation of both FAS and PKS ACPs in vivo. RESULTS: We demonstrate that type II PKS ACPs catalyse self-malonylation upon incubation with malonyl CoA in vitro. The self-malonylation reaction of the actinorhodin C17S holo-ACP has a K(m) for malonyl CoA of 219 microM and a kcat of 0.34 min-1. Complete acylation of the PKS ACPs was observed with malonyl, methylmalonyl and acetoacetyl CoAs. No reaction was observed with acetyl and butyryl CoAs and FAS ACPs did not react with any of the substrates. Recombinant FAS MT from Streptomyces coelicolor did not accelerate the rate of malonylation. CONCLUSIONS: The catalytic self-acylation of type II PKS ACPs is an unprecedented reaction. We propose a reaction mechanism in which conserved arginines form a salt bridge with the acyl moiety and sequester it from bulk solvent. This work suggests that the beta-ketoacyl synthase, chain length factor and ACP may constitute a truly minimal PKS in vivo.

Acyl Carrier Protein↗
PubMed1993

The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli.

Branched-chain keto acid dehydrogenase is a multienzyme complex which is required for the metabolism of the branched-chain amino acids in Pseudomonas putida. The structural genes encoding all four proteins of the bkd operon have been cloned, and their nucleotide sequences have been determined (G. Burns, K. T. Madhusudhan, K. Hatter, and J. R. Sokatch, p. 177-184 in S. Silver, A. M. Chakrabarty, B. Iglewski, and S. Kaplan [ed.], Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology, American Society for Microbiology, Washington D.C., 1990). An open reading frame which encoded a protein with 36.5% amino acid identity to the leucine-responsive regulatory protein (Lrp) of Escherichia coli was found immediately upstream of the bkd operon. Chromosomal mutations affecting this gene, named bkdR, resulted in a loss of ability to use branched-chain amino acids as carbon and energy sources and failure to produce branched-chain keto acid dehydrogenase. These mutations were complemented in trans by plasmids which contained intact bkdR. Mutations affecting bkdR did not have any effect on transport of branched-chain amino acids or transamination. Therefore, the bkdR gene product must affect expression of the bkd operon and regulation must be positive. Mutations affecting bkdR could also be complemented by plasmids containing lrp of E. coli. This is the first instance of a Lrp-like protein demonstrated to regulate expression of an operon outside of E. coli.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2003

Molecular and phenotypic heterogeneity in mitochondrial trifunctional protein deficiency due to beta-subunit mutations.

The mitochondrial trifunctional protein (TFP) is a multienzyme complex of the fatty acid beta-oxidation cycle. It is composed of four alpha-subunits (HADHA) harboring long-chain enoyl-CoA hydratase and long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) and four beta-subunits (HADHB) harboring long-chain 3-ketoacyl-CoA thiolase (LKAT). Mutations in either subunit can result in TFP deficiency with reduced activity of all three TFP enzymes. We characterize 15 patients from 13 families with beta-subunit mutations by clinical, biochemical, and molecular features. Three clinical phenotypes are apparent: a severe neonatal presentation with cardiomyopathy, Reye-like symptoms, and early death (n=4); a hepatic form with recurrent hypoketotic hypoglycemia (n=2); and a milder later-onset neuromyopathic phenotype with episodic myoglobinuria (n=9). Maternal HELLP syndrome occurred in two mothers independently of the fetal phenotype. Mutational analysis revealed 16 different mutations, the majority being missense mutations (n=12). The predominance of missense mutations and the milder myopathic phenotype are consistent. Based upon homology to yeast thiolase that has been characterized structurally, the mutation localization within the protein correlates with the clinical phenotype. Outer loop mutations that are expected to alter protein stability less were only present in milder forms. The degree of reduction in thiolase antigen also correlated with the severity of clinical presentation. Although TFP deficiency is highly heterogeneous, there is genotype-phenotype correlation.

Adolescent↗
PubMed2003

Complete deficiency of mitochondrial trifunctional protein due to a novel mutation within the beta-subunit of the mitochondrial trifunctional protein gene leads to failure of long-chain fatty acid beta-oxidation with fatal outcome.

UNLABELLED: The mitochondrial trifunctional protein (MTP) is a multienzyme complex which catalyses three of the four chain-shortening reactions in the beta-oxidation of long-chain fatty acids. Clinically, failure of long-chain fatty acid beta-oxidation leads to hypoketotic hypoglycaemia associated with coma, hepatopathy, skeletal myopathy and cardiomyopathy. We report on consanguineous parents with six children, four of whom had unexpectedly died in Egypt during the neonatal period due to cardiomyopathy of unknown aetiology and respiratory failure. After moving to Germany, two further children died at the age of 4 months and 12 h, respectively, with signs of respiratory and cardiac failure, hydrops fetalis and acidosis. Analysis of acylcarnitine profiles in dried blood spots of the last two children by electrospray tandem mass spectrometry was indicative of a long-chain fatty acid beta-oxidation disorder. Both infants were homozygous for a novel missense mutation (976G-->C) within a highly conserved region of the MTP beta-subunit gene. Immunoblot analysis in chorionic villi obtained during the subsequent pregnancy demonstrated absence of MTP. In fibroblasts and liver, activities of all three catalytic units of MTP were markedly decreased, further confirming the diagnosis of MTP deficiency. CONCLUSION: the detected mutation (976G-->C) within the beta-subunit of the mitochondrial trifunctional protein gene destabilises the protein, leading to complete deficiency and a poor prognosis. Immunoblot analysis of mitochondrial trifunctional protein in chorionic villi may be a valuable tool for the prenatal diagnosis of the disorder when the molecular genetic defect is unknown.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1990

Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon.

Branched-chain keto acid dehydrogenase is a multienzyme complex produced by Pseudomonas putida when it is grown in a minimal medium containing branched-chain amino acids. A 1.87-kilobase (kb) DNA fragment was cloned and sequenced which contained 0.24 kb of the E1 alpha structural gene and 1.6 kb of upstream DNA. There were 854 base pairs (bp) of noncoding DNA upstream of bkdA1, the first gene of the bkd operon, and 592 bp between the transcriptional and translational starts. The G + C content of the noncoding region was 56.7% compared with 65.2% for all the structural genes of the operon. A partial open reading frame was found on the strand opposite that of the bkd operon beginning at base 774. When the bkd promoter was cloned into the promoter probe vector pKT240, streptomycin resistance was obtained in P. putida but not Escherichia coli with the promoter in both orientations, which indicates either that the bkd promoter is bidirectional or that there are two promoters in this region. A series of ordered deletions on both sides of the proposed site of the start of transcription revealed that almost 700 bp upstream of the start of translation were required for expression. Streptomycin resistance was also obtained in an rpoN mutant of P. putida KT2440 containing constructs with the intact bkd promoter, indicating that the bkd operon does not require the rpoN sigma factor for expression. Another construct containing the bkd promoter, bkdA1, and bkdA2 in pKT240 was used to transform P. putida JS113, a mutant which was unable to produce the E1 subunits of the branched-chain keto acid dehydrogenase. In this case, very high inducible expression of the bkd operon was obtained.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2005

Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase.

Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several alpha-keto acids. In its functional form, it is bound covalently in an amide linkage to the epsilon-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg(-1), indicating a turnover number of approximately 0.2 s(-1), and the apparent Km values for octanoyl-ACP and apo-H protein are 10.2+/-4.4 and 13.2+/-2.9 microM, respectively.

Acyl Carrier Protein↗
PubMed1964

ELECTRON MICROSCOPIC AND BIOCHEMICAL STUDIES OF PYRUVATE DEHYDROGENASE COMPLEX OF ESCHERICHIA COLI.

Examination with the electron microscope of the pyruvate dehydrogenase complex and its component enzymes from Escherichia coli indicates that the complex has a polyhedral structure with a diameter of about 300 to 350 angstroms and a height of 200 to 250 angstroms. The reconstituted complex closely resembles the native complex in appearance. A tentative model of this multienzyme complex is proposed on the basis of the correlated biochemical and electron-microscopic data

Acetates↗
PubMed1998

Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex.

Mitochondrial cytochrome bc1 complex performs two functions: It is a respiratory multienzyme complex and it recognizes a mitochondrial targeting presequence. Refined crystal structures of the 11-subunit bc1 complex from bovine heart reveal full views of this bifunctional enzyme. The "Rieske" iron-sulfur protein subunit shows significant conformational changes in different crystal forms, suggesting a new electron transport mechanism of the enzyme. The mitochondrial targeting presequence of the "Rieske" protein (subunit 9) is lodged between the two "core" subunits at the matrix side of the complex. These "core" subunits are related to the matrix processing peptidase, and the structure unveils how mitochondrial targeting presequences are recognized.

Amino Acid Sequence↗
PubMed2004

Mitochondria, sex, and mortality.

It has been proposed that prior to the evolution of sex, the endosymbiotic relationship between mitochondria and nuclear genomes would have selected mechanisms that maintained the optimum interaction between the two genomes. Once sex evolved, mating would introduce different, competitive, mtDNA and/or nDNA gene products that could well upset the balance. Mechanisms, such as the specific degradation of one mitochondrial genome that is known to occur, could have been selected to prevent part of such competition. Unlike most protein complexes in the cell, the proteins of the multienzyme complexes of the ox-phos system are derived from both nuclear-genome-coded genes and mitochondrial-genome-coded genes. Minor mutations in either mtDNA or nDNA coding for these proteins are known to lead to major and catastrophic diseases of humans, suggesting that very tight and precise interactions are required. To maintain the evolutionarily established balance after mating, monoallelic expression of the nuclear-coded genes would be advantageous and prevent subtly different competitive proteins from interacting with the resident mitochondria. This would require regulation of the expression of those specific nuclear genes, possibly under the control of the resident mitochondria. It is possible that aging cells could lose the requisite tight regulation and allow expression of proteins derived from the formerly repressed nuclear alleles that would compete for mitochondrial complex sites. With age, random failure of this control could lead to increasingly inefficient mitochondria in different tissues and organs and eventually to senescence and death.

Aging↗
PubMed1985

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver.

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.

Ammonium Chloride↗
PubMed1975

X-ray small-angle studies of the pyruvate dehydrogenase core complex from Escherichia coli K-12. I. Overall structure of the core complex.

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex which can be obtained with a unique polypeptide chain composition, has been investigated in solution with the X-ray small-angle technique. The molecular mass of the core complex of 3.78-10(6) daltons verifies the ratio of polypeptide chains of 16:16:16 of the three enzyme components, pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, present in the complex. In connection with the values obtained for the radius of gyration (156.5A), volume (1.07(7) A3) and amount of solvent associated with the complex (1.03 g/g) a loose packing of subunits in the complex has to be assumed. The maximum diameter of the core complex of 433 A, as determined from the correlation function, corroborates the large extension of the complex. The comparison of experimental and theoretical scattering curves reveals a relatively isometric overall shape of the core complex.

Escherichia coli↗