Mutants in Glucose Metabolism

Fraenkel, D G

doi:10.1146/annurev.bi.55.070186.001533

Cited by 50 publications

(36 citation statements)

References 74 publications

(113 reference statements)

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“…This overproduction of NADPH is deleterious, as limited capacity for reoxidation of NADPH is one reason for the low growth rate of phosphoglucose isomerase-deficient E. coli [34]. However, exclusive glucose catabolism via the ED pathway does not support growth of E. coli, as double mutants in both isoforms of phosphofructokinase cannot grow on glucose as the sole carbon source [27].…”

Section: Discussionmentioning

confidence: 99%

“…The PfkA mutant is deficient in the allosterically regulated, major isoform of phosphofructokinase that constitutes about 90% of the total phosphofructokinase activity [27,28]. As phosphofructokinase is required for glucose catabolism via both glycolysis and PP pathway, the very low specific glucose uptake rate of the PfkA mutant and, as a consequence, the low growth rate on glucose are expected (Table 3).…”

Section: Metabolic Flux Ratio Analysis Of E Coli Mutants Of Central mentioning

confidence: 99%

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Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

Fischer

Sauer

2003

European Journal of Biochemistry

348

418

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We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13 C] and [U-13 C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the EntnerDoudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.Keywords: Entner-Doudoroff pathway; flux analysis; fluxome; METAFoR analysis; pentose phosphate pathway.Comprehensive and quantitative understanding of biochemical reaction networks requires not only knowledge about their constituting components, but also information about the behavior of the network in its entirety. Toward this end, systems-oriented methodologies were developed that simultaneously access the level of reaction intermediates [1] or rates of reactions [2][3][4][5], also referred to as the metabolome [6] and the fluxome [7], respectively. The most important property of biochemical networks are the per se nonmeasurable in vivo reaction rates, which may be estimated by so-called metabolic flux analysis that provides a holistic perspective on metabolism.In its simplest form, metabolic flux analysis relies on flux balancing of extracellular consumption and secretion rates within a stoichiometric reaction model [5]. To increase reliability and resolution of such flux balancing analyses, additional information may be derived from 13 C-labeling experiments. In this approach, 13 C-labeled substrates are administered and 13 C-labeled products of metabolism are analyzed by methods that distinguish between different isotope labeling patterns, in particular NMR and MS [2,3,8]. In the most advanced methodology, a comprehensive isotope isomer (isotopomer) model of metabolism is used to map metabolic fluxes in an iterative fitting procedure on the isotopomer pattern of network metabolites that are deduced from NMR or M...

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Section: Discussionmentioning

confidence: 99%

Section: Metabolic Flux Ratio Analysis Of E Coli Mutants Of Central mentioning

confidence: 99%

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

Fischer

Sauer

2003

European Journal of Biochemistry

348

418

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“…18 TIM structure and mechanism have been characterized in great detail19 -25. TIM activity was designed into ecRBP using structure-based computational design techniques to implement a minimalist reaction mechanism9 (Fig.…”

Section: Transplantation Of Novotim Activity From Ecrbp To Tterbpmentioning

confidence: 99%

RETRACTED: Local Encoding of Computationally Designed Enzyme Activity

Allert

Dwyer

Hellinga

2007

Journal of Molecular Biology

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One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously we have shown that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here we report that these mutations can be "transplanted" into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 10 5 -10 6 -fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is locally encoded within the layer of residues directly in contact with the substrate, and that in this case at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants.

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“…Several enzymes of the glycolytic pathway have been cloned and studied on a molecular basis in yeast [5]. However, very little is known about the aldolase reaction in the context of function and regulation of glycolysis and gluconeogenesis and about the interaction of aldolase with other glycolytic enzymes and with membranes (reviewed by Srere [6]).…”

mentioning

confidence: 99%

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae

Schwelberger

Kohlwein

Paltauf

1989

European Journal of Biochemistry

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A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a YeastlEscherichiu coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBAI coding for yeast aldolase. The primary structure of the FBAI gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBAI gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBAI gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBAI allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase alleleJhu1 : : URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.Aldolase (fructose 1,6-bisphosphate aldolase) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate and is present in all animal and plant tissues and most microorganisms [l]. Aldolases of higher organisms belong to class I, which is characterized by its molecular mechanism of cleavage of fructose 1,6-bisphosphate via a covalently linked imino-intermediate [I]. Many class I enzymes of various animals have been analyzed in great detail and DNA sequences of some aldolase genes have been reported recently (see [2] for pertinent references).In contrast to the aldolases in higher organisms, information about the molecular properties of the yeast enzyme is scarce. Richards and Rutter [3] reported the isolation of aldolase from Saccharomyces cerevisiue and identified the enzyme as a class I1 species: Native protein consists of two subunits of 40 kDa, each of which contains one tightly bound zinc cation essential for catalytic function [4].Several enzymes of the glycolytic pathway have been cloned and studied on a molecular basis in yeast [5]. However, very little is known about the aldolase reaction in the context of function and regulation of glycolysis and gluconeogenesis and about the interaction of aldolase with other glycolytic enzymes and with membranes (reviewed by Srere [6]). This prompted us to clone the structural gene of fructose 1,6-bisphosphate aldolase from S . cerevisiae in order to study its regulation of expression and the role of its gene product in the yeast glycolytic pathway in terms of enzyme clust...

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Mutants in Glucose Metabolism

Cited by 50 publications

References 74 publications

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

RETRACTED: Local Encoding of Computationally Designed Enzyme Activity

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae

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