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PubMed2003

Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver.

While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH13CO3, [U-13C3]propionate, or [U-13C3]lactate + [U-13C3]pyruvate, under various concentrations of propionate. The mass isotopomer distributions of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA revealed that, in intact livers and hearts, (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA racemase reaction keeps the methylmalonyl-CoA enantiomers in isotopic equilibrium under all conditions tested, and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flow of propionyl-CoA increases. The thermodynamic dis-equilibrium of the combined reactions of the propionyl-CoA pathway explains the effectiveness of anaplerosis from propionyl-CoA precursors such as heptanoate.

Acyl Coenzyme A↗

PubMed2004

Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases.

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.

Amino Acid Sequence↗

PubMed2004

The cblD defect causes either isolated or combined deficiency of methylcobalamin and adenosylcobalamin synthesis.

Intracellular cobalamin is converted to adenosylcobalamin, coenzyme for methylmalonyl-CoA mutase and to methylcobalamin, coenzyme for methionine synthase, in an incompletely understood sequence of reactions. Genetic defects of these steps are defined as cbl complementation groups of which cblC, cblD (described in only two siblings), and cblF are associated with combined homocystinuria and methylmalonic aciduria. Here we describe three unrelated patients belonging to the cblD complementation group but with distinct biochemical phenotypes different from that described in the original cblD siblings. Two patients presented with isolated homocystinuria and reduced formation of methionine and methylcobalamin in cultured fibroblasts, defined as cblD-variant 1, and one patient with isolated methylmalonic aciduria and deficient adenosylcobalamin synthesis in fibroblasts, defined as cblD-variant 2. Cell lines from the cblD-variant 1 patients clearly complemented reference lines with the same biochemical phenotype, i.e. cblE and cblG, and the cblD-variant 2 cell line complemented cells from the mutant classes with isolated deficiency of adenosylcobalamin synthesis, i.e. cblA and cblB. Also, no pathogenic sequence changes in the coding regions of genes associated with the respective biochemical phenotypes were found. These findings indicate heterogeneity within the previously defined cblD mutant class and point to further complexity of intracellular cobalamin metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2005

Mechanistic insights into the isochorismate pyruvate lyase activity of the catalytically promiscuous PchB from combinatorial mutagenesis and selection.

PchB from Pseudomonas aeruginosa possesses isochorismate pyruvate lyase (IPL) and weak chorismate mutase (CM) activity. Homology modeling based on a structurally characterized CM, coupled with randomization of presumed key active site residues (Arg54, Glu90, Gln91) and in vivo selection for CM activity, was used to derive mechanistic insights into the IPL activity of PchB. Mutation of Arg54 was incompatible with viability, and the CM and IPL activities of an engineered R54K variant were reduced 1,000-fold each. The observation that position 90 was tolerant to substitution but position 91 was essentially confined to Gln or Glu in functional variants rules out involvement of Glu90 in general base catalysis. Counter to the generally accepted mechanistic hypothesis for pyruvate lyases, we propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs. A common catalytic principle for both PchB functions is also supported by the covariance of the catalytic parameters for the CM and IPL activities and the shared functional requirement for a protonated Glu91 in Q91E variants. The experiments demonstrate that focusing directed evolution strategies on the readily accessible surrogate activity of an enzyme can provide valuable insights into the mechanism of the primary reaction.

Amino Acid Sequence↗

PubMed2005

An active enzyme constructed from a 9-amino acid alphabet.

Nature employs a set of 20 amino acids to produce a repertoire of protein structures endowed with sophisticated functions. Here, we combined design and selection to create an enzyme composed entirely from a set of only 9 amino acids that can rescue auxotrophic cells lacking chorismate mutase. The simplified protein captures key structural features of its natural counterpart but appears to be somewhat less stable and more flexible. The potential of a dramatically reduced amino acid alphabet to produce an active catalyst supports the notion that primordial enzymes may have possessed low amino acid diversity and suggests that combinatorial engineering strategies, such as the one used here, may be generally applied to create enzymes with novel structures and functions.

Amino Acid Sequence↗

PubMed2003

Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia.

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites. Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis. Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process. The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor. Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA. Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated. However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high. In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation. Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity. The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L. in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity. All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1994

Cobalt-vitamin B12 deficiency causes accumulation of odd-numbered, branched-chain fatty acids in the tissues of sheep.

Nine 5-month-old lambs were randomly allocated to two groups and were fed on either a Co-deficient whole-barley diet (n5), or the same diet supplemented with Co (n4). The lambs were fed on their respective diets for 28 weeks. Plasma vitamin B12 concentrations fell below the lower limit of normality after 6 weeks, and plasma methylmalonic acid (MMA) concentrations rose above the upper limit of normality after 10 weeks. However, plasma MMA concentrations fell to near normal levels towards the end of the experiment suggesting that diagnosis of more severe Co deficiency based on determination of plasma MMA concentrations may be of limited value. Analysis of tissue samples collected at slaughter revealed a marked reduction in the vitamin B12 concentration and the activity of methylmalonyl-CoA mutase (EC5.4.99.2) in the tissues taken from the Co-deficient sheep, by comparison with the controls. Although tissue concentrations of MMA in the Co-deficient animals were not significantly different from those of the controls, we did detect increased concentrations of branched-chain fatty acids. This suggested that misincorporation of MMA, but not propionic acid, into fatty acids had occurred. The Co-deficient lambs did not develop any neurological signs, suggesting that accumulation of branched-chain fatty acids may not be involved in the development of neurological lesions.

Animals↗

PubMed1981

A simple procedure for the isolation of seven abundant muscle enzymes.

The present work describes procedures in which seven major muscle enzymes and serum albumin can be simultaneously isolated from chicken skeletal muscles. The seven enzymes isolated were: phosphorylase, enolase, creatine-P kinase, aldolase, glyceraldehyde-3-P dehydrogenase, phosphoglycerate mutase, and triose-P isomerase. The proteins isolated by these methods were judged to be greater than 97% pure on the basis of electrophoretic analysis in sodium dodecyl sulfate polyacrylamide gels. The procedure is applicable for isolation of the enzymes from large (greater than 100 g) or small (less than 0.5 g) amounts of muscle tissue and the entire procedure can be completed within two days. Particularly useful features of the procedures are: (1) preferential solubilization of the enzymes from myofibrils by extraction of muscle specimens in solutions of different ionic strength; (2) specific precipitation of phosphorylase, creatine-P kinase, and glyceraldehyde 3-Phosphate dehydrogenase from solutions of specified pH and degrees of ammonium sulfate saturation; and (3) an alternate method for isolation of glyceraldehyde-3-P dehydrogenase by specific elution of the enzyme from phosphocellulose columns with ATP. Because of the ease, rapidity, and reproducibility of the procedures, these methods may be useful for the routine isolation of the muscle enzymes in studies on biochemical regulation, as well as for obtaining large quantitites of the enzymes for structural analysis.

Animals↗

PubMed1987

High performance purification of glycolytic enzymes and creatine kinase from chicken breast muscle and preparation of their specific immunological probes.

We developed a novel procedure for isolation of the muscle isozymes of aldolase, triose phosphate isomerase (TPI), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase, pyruvate kinase (PK) and lactic dehydrogenase (LDH), and also creatine kinase (CK), at high purity, specific activity and yield. Protein was extracted from chicken breast muscle and glycolytic enzymes were purified by a three step procedure consisting of: Ammonium sulfate combined with pH fractionation. Phosphocellulose chromatography with performance of high pressure liquid chromatography, exploiting a pH gradient formed by a gradient of the buffering ion for protein elution. Affinity chromatography causing elution by substrate or pH. The enzymes, obtained at over 95% purity as judged by specific activity and silver stained electropherograms, were injected into sheep. Antibody for each enzyme was purified on specific immunosorbant and its specificity was verified by immunotransfer analysis.

Animals↗

PubMed2001

A metabolic node in action: chorismate-utilizing enzymes in microorganisms.

The shikimate pathway has been described as a metabolic tree with many branches that led to the synthesis of an extensive range of products. This pathway is present only in bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical reactions of the pathway, significant differences exist in terms of organization and regulation. In the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores. Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase, anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene structures and regulation, nucleotide and protein sequences, protein structures, and reaction mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways. Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence similarity, suggesting divergent evolution and commonality in reaction mechanisms. However, other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction capabilities.

Anthranilate Synthase↗

PubMed1991

Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide.

Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.

Amino Acid Sequence↗

PubMed1997

The aroQ and pheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison.

The gene (denoted aroQp.pheA) encoding the bifunctional P-protein (chorismate mutase-P/prephenate dehydratase) from Xanthomonas campestris was cloned. aroQp.pheA is essential for L-phenylalanine biosynthesis. DNA sequencing of the smallest subclone capable of functional complementation of an Escherichia coli phenylalanine auxotroph revealed a putative open reading frame (ORF) of 1200 bp that would encode a 43,438-Da protein. AroQp.PheA exhibited 51% amino acid identity with a Pseudomonas stutzeri homologoue and greater than 30% identities with AroQp.PheA proteins from Haemophilus influenzae, Neisseria gonorrhoeae, and a number of enteric bacteria. AroQp.PheA from X. campestris, when expressed in E. coli, possesses a 40-residue amino-terminal extension that is lysine-rich and that is absent in all of the AroQp.PheA homologues known at present. About 95% of AroQp.PheA was particulate and readily sedimented by low-speed centrifugation. Soluble preparations of cloned AroQp.PheA exhibited a native molecular mass of 81,000 Da, indicating that the active enzyme species is a homodimer. These preparations were unstable after purification of about 40-fold, even in the presence of glycerol, which was an effective protectant before fractionation. When AroQp.PheA was overproduced by a T7 translation vector, unusual inclusion bodies having a macromolecular structure consisting of protein fibrils were observed by electron microscopy. Insoluble protein collected at low-speed centrifugation possessed high catalytic activity. The single band obtained via SDS-PAGE was used to confirm the translational start via N-terminal amino acid sequencing. A perspective on the evolutionary relationships of monofunctional AroQ and PheA proteins and the AroQp.PheA family of proteins is presented. A serC gene located immediately upstream of X. campestris aroQp.pheA appears to reflect a conserved gene organization, and both may belong to a single transcriptional unit.

Allosteric Regulation↗

PubMed1979

Lack of effect of riboflavin deficiency on vitamin B12-related metabolic pathways and fatty acid synthesis.

The neurological sequelae of riboflavin deficiency posed the possibility that this tissue injury was mediated by defective vitamin B12 function due to the requirement for riboflavin-dependent oxidoreductase systems in B12 coenzyme synthesis and function. Studies of the B12-dependent enzymatic reactions (5-methyltetrahydrofolic-homocysteine methyltransferase and methylmalonyl coenzyme A mutase) in a fiboflavin-deficient rat model documented normal B12 activity in liver and neural tissue. In addition, examination of neural lipids and separation and analysis of neural fatty acids failed to reveal the increased odd chain fatty acids characteristically seen in the B12-deficient state. Thus, the neural tissue sequelae of riboflavin deficiency do not appear to relate to B12 coenzyme function.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1980

Effect of dietary and nitrous oxide-induced vitamin B-12 deficiency on uptake of 5-methyltetrahydrofolate by isolated rat hepatocytes.

Prior exposure of rats to a mixture of nitrous oxide/oxygen (80/20) for 20 to 24 hours and feeding rats a diet deficient in vitamin B-12 and methionine for 23 weeks resulted in a 86 to 90% decrease in the activity of the vitamin B-12 dependent enzyme, 5-methyltetrahydrofolate-homocysteine methyltransferase (EC 4.2.99.10). In addition, deficient animals excreted some 15 times as much methylmalonic acid as controls indicating impaired functioning of the vitamin B-12-dependent enzyme, methylmalonyl CoA mutase (EC 5.4.99.2). Hepatocytes prepared from rats deficient in vitamin B-12 and methionine and from nitrous oxide-treated rats showed no impairment of the membrane transport of 5-methyltetrahydro-[G-3H]folic acid when compared to the respective controls. This observation was true both for the initial rate of entry and for the ability of these cells to accumulate this folate derivative. These findings support the "methyl trap" hypothesis and apparently rule out the "cellular transport" hypothesis for the folate-vitamin B-12 metabolic interrelationship.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1983

Effect of a vitamin B-12-deficient diet on lipid and fatty acid composition of spinal cord myelin in the fruit bat.

The effects of vitamin B-12 deficiency on lipids and fatty acids of spinal cord myelin were studied in control and vitamin B-12-deficient fruit bats. Very low plasma and brain vitamin B-12 concentrations were present in animals fed the vitamin B-12-free, all fruit diet. Myelin was isolated from the spinal cord of control and vitamin B-12-deficient animals (n = 3 pools) by means of a flotation method in a discontinuous sucrose gradient. The molar concentration of cholineglycerophosphatide was significantly lower in the deficient bats than in the controls. The molar cholesterol: phospholipid ratio in the deficient bats was also significantly lower. The odd-chain fatty acid 15:1 was not detected in cholineglycerophosphatide from the controls, but comprised 1.4% of the total fatty acids in the deficient group. The odd-chain fatty acids 15:0, 17:1 and 19:0 were present in slightly higher amounts in cholineglycerophosphatide of deficient bats. Lipid and fatty acid differences were related to the vitamin B-12-requiring methionine synthetase and methylmalonyl CoA mutase reactions.

Animals↗

PubMed1999

Identification of differentially expressed mRNAs in human fetal liver across gestation.

Differential gene expression, with its precise start and stop times, is believed to be critical for the programmed development of new cells and tissues. Within the developing fetus, one tissue of particular interest is fetal liver. This organ undergoes rapid changes in the pathway toward liver development in utero since it is also the major site of hematopoiesis, until bone marrow hematopoiesis predominates. Believing that patterns would emerge from the bi-weekly large-scale inspection of expressed genes in the fetal liver, we employed differential display reverse transcription-polymerase chain reaction (DDRT-PCR) as ourprimary inspection tool. Using DDRT-PCR, we isolated cDNAs differentially expressed throughout fetal liver development and in adult liver. We displayed approximately 25 000 cDNAs from 10 and 24 week fetal liver and adult liver. From this initial screen, we determined that approximately 0.1-1% of the mRNA population undergoes expression changes. We extracted, purified and sequenced 25 differentially displayed cDNA bands. Fourteen cDNAs had similarities to known genes, while 11 cDNAs were not similar to any characterized gene. The differentially expressed cDNAs from known genes present in fetal liver include alpha-fetoprotein, stem cell factor, erythroid alpha-spectrin, 2,3-bisphosphoglycerate mutase, insulin-like growth factor-2, porphobilinogen deaminase and Mac30. The differentially expressed cDNAs present in adult liver but not in 10 week fetal liver were nicotinamide deaminase, human fibrinogen-related protein and alpha-acid glycoprotein. The majority of differentially expressed genes found during this effort appear to be turned on during organogenesis, however, some genes were found that are apparently turned off completely.

Adult↗

PubMed2005

Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants.

Ozone (O3), a major photochemical oxidant, induces leaf injury concomitant with salicylic acid (SA) synthesis. In pathogen-infected leaves, SA is synthesized via two pathways, involving phenylalanine or isochorismate. SA biosynthesis under O3 fumigation is not well understood. When we applied 14C-labeled benzoic acid (a precursor of SA in the pathway via phenylalanine) to O3-exposed tobacco leaves, it was effectively metabolized to SA. However, the activity and mRNA level of isochorismate synthase (ICS) were not increased. In contrast, ICS activity was increased in O3-exposed Arabidopsis thaliana L. These results suggest that SA is synthesized via benzoic acid from phenylalanine in O3-exposed tobacco leaves but via isochorismate in Arabidopsis. Ethylene is a plant hormone that promotes leaf damage in O3-exposed plants. During O3 exposure, transgenic plants with a phenotype of reduced O3-induced ethylene production accumulated less SA than did wild-type plants. O3 increased the activity of phenylalanine ammonia-lyase (PAL) and the transcript levels of the chorismate mutase (CM) and PAL genes in wild-type tobacco, but their induction was suppressed in the transgenic plants. These results indicate that ethylene promotes SA accumulation by regulating the expression of the CM and PAL genes in O3-exposed tobacco.

Amino Acid Sequence↗

PubMed1998

Molecular shape comparisons in searches for active sites and functional similarity.

Here we examine the reliability of surface comparisons in searches for active sites in proteins. Detection of a patch of surface on one protein which is similar to an active site in another, may suggest similarities in enzymatic mechanisms, in enzyme functions and implicate a potential target for ligand/inhibitor design. Specifically, we compare the efficacy of molecular surface comparisons with comparisons of surface atoms and of C(alpha) backbone atoms. We further investigate comparisons of specific atoms, belonging to a predefined pattern of catalytic residues versus comparisons of molecular surfaces and, separately, of surface atoms. This aspect is particularly relevant, as catalytic residues may be (partially) buried. We also explore active site comparisons versus comparisons in which the entire molecular surfaces are scanned. While here we focus on the geometrical aspect of the problem, we also investigate the effect of adding residue labels in these comparisons. Our extensive studies cover the serine proteases, containing the highly conserved triad motif, and the chorismate mutases. Since such active site comparisons entail comparisons between unconnected points in 3D space, an order-independent comparison technique is necessary. The geometric hashing algorithm is ideally suited to handling such a task. It can perform both global shape matching for the whole surfaces of large protein molecules and searching for local shape similarities for small surface motifs. Our results show that molecular surface comparisons work best when the similarity is high. As the similarity deteriorates, the number of potential solutions increases rapidly, making their ranking difficult, particularly when scanning entire molecular surfaces. Utilizing atomic coordinates directly appears more adequate under such circumstances.

Binding Sites↗