Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli.
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Wild-type Escherichia coli K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (-100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (-320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis. (+info)
Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates.
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Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase. (+info)
A family of highly conserved glycosomal 2-hydroxyacid dehydrogenases from Phytomonas sp.
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Phytomonas sp. contains two malate dehydrogenase isoforms, a mitochondrial isoenzyme with a high specificity for oxaloacetate and a glycosomal isozyme that acts on a broad range of substrates (Uttaro, A. D., and Opperdoes, F.R. (1997) Mol. Biochem. Parasitol. 89, 51-59). Here, we show that the low specificity of the latter isoenzyme is the result of a number of recent gene duplications that gave rise to a family of glycosomal 2-hydroxyacid dehydrogenase genes. Two of these genes were cloned, sequenced, and overexpressed in Escherichia coli. Although both gene products have 322 amino acids, share 90.4% identical residues, and have a similar hydrophobicity profile and net charge, their kinetic properties were strikingly different. One isoform behaved as a real malate dehydrogenase with a high specificity for oxaloacetate, whereas the other showed no activity with oxaloacetate but was able to reduce other oxoacids, such as phenyl pyruvate, 2-oxoisocaproate, 2-oxovalerate, 2-oxobutyrate, 2-oxo-4-methiolbutyrate, and pyruvate. (+info)
From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity.
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Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases. (+info)
Reversible inactivation of the isocitrate dehydrogenase of Escherichia coli ML308 during growth on acetate.
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During aerobic growth of Escherichia coli ML308 on acetate as sole carbon source, the apparent synthesis of isocitrate dehydrogenase was repressed relative to cultures on other carbon sources, such as glucose, which do not employ the glyoxylate bypass as an anaplerotic sequence. When cells were removed from an acetate medium, or when compounds were added which made the operation of the glyoxylate bypass unnecessary, the activity of isocitrate dehydrogenase rapidly increased 3- to 4-fold but fell again on restoration to an acetate medium. Changes in activity were rapid and, furthermore, could be demonstrated in the absence of protein synthesis. It is thus improbable that the mechanism involved degradation or de novo synthesis of the enzyme protein. Oxaloacetate and glyoxylate showed concerted inhibition of isocitrate dehydrogenase which could be relieved by dialysis. Because extracts of low enzyme activity, derived from acetate-metabolizing cells, could not be stimulated by dialysis or by addition of a wide range of metabolites, it is unlikely that low molecular weight, freely dissociable effectors were responsible for stimulation or inhibition of activity. Control of isocitrate dehydrogenase permitted the efficient utilization of acetate as sole source of carbon and energy but perserved the capacity of the cell to respond rapidly to an improvement in nutritional conditions. A limited survey showed that the mechanism is common but not universal among strains of E. coli and occurs in at least one strain each of Klebsiella aerogenes, Salmonella typhimurium and Serratia marcescens. (+info)
In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum.
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The C(3)-C(4) metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C(3) carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C(4)-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a (13)C NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a (13)C-labeled glucose isotopomer mixture as the main carbon source and (13)C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7% to C(3) carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C(4) metabolites for biosynthesis. (+info)
Self-organizing biochemical cycles.
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I examine the plausibility of theories that postulate the development of complex chemical organization without requiring the replication of genetic polymers such as RNA. One conclusion is that theories that involve the organization of complex, small-molecule metabolic cycles such as the reductive citric acid cycle on mineral surfaces make unreasonable assumptions about the catalytic properties of minerals and the ability of minerals to organize sequences of disparate reactions. Another conclusion is that data in the Beilstein Handbook of Organic Chemistry that have been claimed to support the hypothesis that the reductive citric acid cycle originated as a self-organized cycle can more plausibly be interpreted in a different way. (+info)
Different patterns of energy metabolism in the rat and mouse zygate.
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The development of rat zygotes in vitro to the two-cell stage occurred if lactate, phosphoenolpyruvate (PEP), pyruvate or oxaloacetate were present in the media. When rat and mouse zygotes were cultured in the same droplet of medium containing lactate or PEP, mouse zygotes did not develop to the two-cell stage but the rat zygotes cleaved. (+info)