Enzyme induction and repression in Arthrobacter crystallopoietes

Summary Catabolic effects which exert control over the inducible synthesis of three enzymes in Arthrobacter crystallopoietes involve at least three different mechanisms: interference with inducer transport, severe catabolite repression, and transient repression. The rate of histidase induction by histidine is reduced by incubation of the cells with succinate or glucose. The maximum effect of succinate, 67% reduction in histidase production, occurs only after 100 min of incubation with succinate. At least 3h of incubation are required for the maximum effect of glucose (31% reduction in enzyme induction). Both succinate and glucose inhibit histidine transport. Cyclic adenosine 3,5-monophosphate (cyclic AMP), at 10^-7 M, slightly stimulates the induction of histidase in cultures both with or without succinate. No conditions were found in which cyclic AMP abolishes the effect of succinate. Induction of l-serine dehydratase by glycine is severely and permanently repressed by glucose and to a lesser extent by citrate. Glucose does not affect glycine uptake. Succinate, fumarate, and aspartate, which are all better substrates than glucose or citrate for growth of A. crystallopoietes, have no effect on l-serine dehydratase induction. Induction and repression of l-serine dehydratase are not affected by cyclic AMP. Synthesis of isocitrate lyase after addition of acetate is unaffected by glucose but is severely repressed by succinate or fumarate. Aspartate and glutamate cause a transient repression of enzyme synthesis after which synthesis proceeds at the control rate. The ability to transport acetate is inducible. Development of this capacity in the presence of acetate is not affected by succinate or glutamate. Cyclic AMP has no effect on enzyme production or repression. A. crystallopoietes takes up radioactive cyclic AMP and has at least one of the enzymes of cyclic AMP metabolism, adenyl cyclase.     

Distinct pathways regulate facilitated glucose transport in human articular chondrocytes during anabolic and catabolic responses

Articular cartilage is an avascular, non-insulin-sensitive tissue that utilizes glucose as the main energy source, a precursor for glycosaminoglycan synthesis, and a regulator of gene expression. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. Previously, we demonstrated that glucose transport in chondrocytes is regulated by proinflammatory cytokines via upregulation of GLUT mRNA and protein expression. The objective of the present study was to determine differences in molecular mechanisms regulating glucose transport in chondrocytes stimulated with the anabolic transforming growth factor-beta1 (TGF-beta1) vs. the catabolic and proinflammatory cytokine IL-1beta. Both TGF-beta1 and IL-1beta accelerate glucose transport in chondrocytes. Although both IL-1beta and TGF-beta1 enhance glucose transport in chondrocytes to a similar magnitude, IL-1beta induces significantly higher levels of lactate. TGF-beta1-stimulated glucose transport is not associated with increased expression or membrane incorporation of GLUT1, -3, -6, -8, and -10 and depends on PKC and ERK activation. In contrast, IL-1beta-stimulated glucose transport is accompanied by increased expression and membrane incorporation of GLUT1 and -6 and depends upon activation of PKC and p38 MAP kinase. In conclusion, anabolic and catabolic stimuli regulate facilitated glucose transport in human articular chondrocytes via different effector and signaling mechanisms, and they have distinct effects on glycolysis.     

Junctions of catabolic and anabolic pathways in Zymomonas mobilis: phosphoenolpyruvate carboxylase and malic enzyme

Summary The synthesis of oxalacetate and malate in the ethanol-producing bacterium Zymomonas mobilis have been investigated. Cell-free extracts were examined for pyruvate carboxylase, phosphoenolpyruvate (PEP) carboxylase, PEP carboxytransphosphorylase, PEP carboxykinase, and malic enzyme, but only PEP carboxylase and nicotine adenine dinucleotide (NAD)-dependent malic enzyme activities could be detected. The PEP carboxylase, partially purified from extracts, was not affected by acetyl-coenzyme A. Intermediates of the tricarboxylic acid cycle and aspartate inhibited the enzyme competitively with PEP. Of these, citrate and -ketoglutarate were the strongest inhibitors. The physiological roles of PEP carboxylase and malic enzyme in Z. mobilis are discussed.Dedicated to Prof. Dr. A. Fiechter, ETH Zürich, on the occasion of his 65th birthday     

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.     

Stereospecificity in meta-fission catabolic pathways

The stereospecificities of 2-keto-4-pentenoate hydratase and 4-hydroxy-2-ketovalerate aldolase were studied in Escherichia coli, Pseudomonas putida, and Acinetobacter sp. Hydration was stereospecific in all three; however, only P. putida and Acinetobacter sp. showed stereospecificity in their aldolase reactions.     

The earliest catabolic pathways

Summary Anaerobic catabolism of amino acids may have provided the main source of energy for primitive microorganisms. Examples are given of amino acid catabolic reactions coupled to substrate level phosphorylations occurring in present-day anaerobes which may be biochemical fossils from a very early stage of the evolution of procaryotes.     

Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme

Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase). In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties. The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity. The structural basis for this kinetic specialization was examined. The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired. Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.     

The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways

Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.     

Evolution of chlorocatechol catabolic pathways

The aerobic bacterial degradation of chloroaromatic compounds often involves chlorosubstituted catechols as central intermediates. They are converted to 3-oxoadipate in a series of reactions similar to that for catechol catabolism and therefore designated as modifiedortho-cleavage pathway. Among the enzymes of this catabolic route, the chlorocatechol 1,2-dioxygenases are known to have a relaxed substrate specificity. In contrast, several chloromuconate cycloisomerases are more specific, and the dienelactone hydrolases of chlorocatechol catabolic pathways do not even convert the corresponding intermediate of catechol degradation, 3-oxoadipate enol-lactone. While the sequences of chlorocatechol 1,2-dioxygenases and chloromuconate cycloisomerases are very similar to those of catechol 1,2-dioxygenases and muconate cycloisomerases, respectively, the relationship between dienelactone hydrolases and 3-oxoadipate enol-lactone hydrolases is more distant. They seem to share an / hydrolase fold, but the sequences comprising the fold are quite dissimilar. Therefore, for chlorocatechol catabolism, dienelactone hydrolases might have been recruited from some other, preexisting pathway. Their relationship to dienelactone (hydrolases identified in 4-fluorobenzoate utilizing strains ofAlcaligenes andBurkholderia (Pseudomonas) cepacia is investigated). Sequence evidence suggests that the chlorocatechol catabolic operons of the plasmids pJP4, pAC27, and pP51 have been derived from a common precursor. The latter seems to have evolved for the purpose of halocatechol catabolism, and may be considerably older than the chemical industry.     

Staphylococcal L-asparaginase: catabolic repression of synthesis.

The regulation mechanism of production of staphylococcal L-asparaginase was investigated. The role of cAMP in regulation of its synthesis was confirmed. Production of L-asparaginase from S. aureus NCTC 4135 was inhibited by all carbon sources, mono- and disaccharides added to the growth medium. The strongest inhibition was caused by saccharose and maltose, whereas weaker by galactose, lactose, mannitol and mannose. It was found that exogenous cAMP in the presence of carbon sources stimulated synthesis of the investigated enzyme.     

Laboratory evolution of catabolic enzymes and pathways

The laboratory evolution of environmentally relevant enzymes and proteins has resulted in the generation of optimized and stabilized enzymes, as well as enzymes with activity against new substrates. Numerous methods, including random mutagenesis, site-directed mutagenesis and DNA shuffling, have been widely used to generate variants of existing enzymes. These evolved catabolic enzymes have application for improving biodegradation pathways, generating engineered pathways for the degradation of particularly recalcitrant compounds, and for the development of biocatalytic processes to produce useful compounds. Regulatory proteins associated with catabolic pathways have been utilized to generate biosensors for the detection of bioavailable concentrations of environmentally relevant chemicals.     

Cellular distribution of ornithine in Neurospora: anabolic and catabolic steady states.

During growth on minimal medium, cells of Neurospora contain three pools of ornithine. Over 95% of the ornithine is in a metabolically inactive pool in vesicles, about 1% is in the cytosol, and about 3% is in the mitochondria. By using a ureaseless strain, we measured the rapid flux of ornithine across the membrane boundaries of these pools. High levels of ornithine and the catabolic enzyme ornithine aminotransferase coexist during growth on minimal medium but, due to the compartmentation of the ornithine, only 11% was catabolized. Most of the ornithine was used for the synthesis of arginine. Upon the addition of arginine to the medium, ornithine was produced catabolically via the enzyme arginasn early enzyme of ornithine synthesis. The biosynthesis of arginine itself, from ornithine and carbamyl phosphate, was halted after about three generations of growth on arginine via the repression of carbamyl phosphate synthetase A. The catabolism of arginine produced ornithine at a greater rate than it had been produced biosynthetically, but this ornithine was not stored; rather it was catabolized in turn to yield intermediates of the proline pathway. Thus, compartmentation, feedback inhibition, and genetic repression all play a role to minimize the simultaneous operation of anabolic and catabolic pathways for ornithine and arginine.     

Ammonium and glucose repression of the arginine catabolic enzymes in Aspergillus nidulans

Summary The synthesis of two enzymes of the arginine catabolic pathway, arginase and ornithine -transaminase (OTAse), in Aspergillus nidulans was found to be sensitive to both glucose and ammonium repression. The glucose and nitrogen starvation result in the identical derepression of OTAse synthesis and have no effects on arginase synthesis. Glucose and ammonium affect the kinetics of induction of both enzymes, however, the effect of ammonium is much stronger. Evidence was obtained for the direct involvement of ammonium in the repression phenomenon. The relations between glucose and ammonium repression are discussed.