2-ketobutyrate: A putative alarmone of Escherichia coli

Daniel, Jacques; Dondon, L.; Danchin, Antoine

doi:10.1007/bf00331076

Cited by 32 publications

(19 citation statements)

References 22 publications

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“…5C), which increases its production, and by L-valine inhibition of the AHAS I and AHAS III isozymes, which reduces its consumption. It is now known that this ␣KB accumulation is toxic to cells because of its ability to inhibit the glucose PTS transport system (24,25). Thus, as reproduced by our simulations, L-valine growth inhibition of E. coli K12 is not a consequence of L-isoleucine starvation.…”

Section: Responses To Metabolic and Genetic Perturbations L-valine Grsupporting

confidence: 66%

“…However, concomitant with the decrease in ␣KB observed in the presence of 500 M L-isoleucine, the cellular TDA in the active R state is reversed to the control level observed in the absence of L-valine. These simulations are verified by experimental results accumulated from multiple laboratories over a three decade period (6,24,25).…”

Section: Responses To Metabolic and Genetic Perturbations L-valine Grsupporting

confidence: 63%

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A Mathematical Model for the Branched Chain Amino Acid Biosynthetic Pathways of Escherichia coli K12

Yang

Shapiro

Hung

et al. 2005

Journal of Biological Chemistry

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Systems biology may be broadly defined as the integration of diverse data into useful biological models that allow scientists to easily observe complex cellular behaviors and predict the outcomes of metabolic and genetic perturbations. As a first step toward the elucidation of the systems biology of the model organism Escherichia coli, we have elected to limit our initial efforts to the development of a mathematical model for the complex but well studied metabolic pathways for the biosynthesis of the branched chain amino acids L-isoleucine, L-valine, and L-leucine.The biosynthetic pathways for the branched chain amino acids are shown in Fig. 1 (1-3). L-Threonine deaminase (TDA), 1 the first enzyme specific for the biosynthesis of L-isoleucine, is end product-inhibited by L-isoleucine, and ␣-isopropylmalate synthase (IPMS), the first enzyme specific for the biosynthesis of L-leucine, is end product-inhibited by L-leucine. However, because the parallel pathways for L-valine and L-isoleucine biosynthesis are catalyzed by a set of bi-functional enzymes that bind substrates from either pathway, L-valine inhibition of the first enzyme specific for its biosynthesis catalyzed by a single ␣-acetohydroxy acid synthase (AHAS) could compromise the cell for L-isoleucine biosynthesis or result in the accumulation of a toxic metabolic intermediate, ␣-ketobutyrate (␣KB). This type of regulatory problem is often solved by using multiple isozymes with different substrate preferences that are differentially regulated by multiple end products of parallel pathways. In this case, there are three AHAS isozymes that catalyze the first step of the L-valine and the second step of the L-isoleucine pathway (4). AHAS I has substrate preferences for the condensation of two pyruvate molecules required for Lvaline biosynthesis and is end product-inhibited by L-valine (4). AHAS III shows no preference for pyruvate or ␣KB. Although this isozyme can produce intermediates for both L-valine and L-isoleucine, it is inhibited by L-valine (4). The AHAS II isozyme has substrate preferences for the condensation of pyruvate and the ␣KB required for L-isoleucine biosynthesis, and it is not inhibited by any of the branched chain amino acids (4). However, AHAS II is not active in the E. coli. K12 strain (5). Consequently, this strain cannot grow in the presence of high levels of L-valine unless L-isoleucine is also added to the growth medium (6).TDA is an allosteric enzyme whose kinetic behavior can be described by the concerted allosteric transition mode of the Monod, Wyman, and Changeux (MWC) model (7,8). According to the MWC model, TDA can exist in an active state (R) or an inactive state (T) (8, 9). The fraction of active enzyme in the R or T states is determined by the concentrations and relative affinities of the substrate (L-threonine), the inhibitor (L-isoleucine), and the activator (L-valine) for the R and T states.

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Section: Responses To Metabolic and Genetic Perturbations L-valine Grsupporting

confidence: 66%

Section: Responses To Metabolic and Genetic Perturbations L-valine Grsupporting

confidence: 63%

A Mathematical Model for the Branched Chain Amino Acid Biosynthetic Pathways of Escherichia coli K12

Yang

Shapiro

Hung

et al. 2005

Journal of Biological Chemistry

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“…The accumulation of threoninederived AKB was monitored by using a minor modification of the radiochemical method of Daniel et al (9). Portions TR5654 TR5655 TR5655 TR5657 TR5658 TR5660 TR5662 TR5663 TR5664 TR5665 TR5666 TR5667 TR5668 TR5669 TR5670 TR5671 TT627 TT628 TT629 TT6197 TV041 TV062 TV063 TV064 TV065 TV066 TV7542.3 E. coli CY307 DG30 MC4100 MM294(pPB13) TV1000 TV1002 TV1006 (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Exogenously added AKB is toxic to S. typhimurium (25,28), although the precise nature of toxicity is unclear. It has been proposed that AKB interferes with valine biosynthesis (28), formation of valyl-tRNAval and isoleucyl-tRNAIle (30), pantothenate biosynthesis (25), pts-mediated sugar transport (9,10), flux to and through the tricarboxylic acid cycle (8), and formation of aspartate (9). It has also been suggested that AKB functions as an alarmone which signals a shift from an anaerobic environment to an aerobic environment (9).…”

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confidence: 99%

Sensitivity of a Salmonella typhimurium aspC mutant to sulfometuron methyl, a potent inhibitor of acetolactate synthase II

Dyk¹,

LaRossa²

1986

J Bacteriol

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Sulfometuron methyl is a potent and specific inhibitor of acetolactate synthase II in Salmonella typhimurium.Mutant strains sensitive to sulfometuron methyl on minimal medium were isolated following mutagenesis with TnlO. A conditionally auxotrophic insertion mutant, strain SMS409, which required aspartate at high temperatures or in the presence of tyrosine, was found among the 15 mutants isolated. The TnWO insertion in strain SMS409 was mapped by conjugation and transduction to the region between aroA and pncB at 20 min on the chromosome of S. typhimurium; this location is similar to the genetic location of aspC in Escherichia coli.The specific activity of the aspC product, aspartate aminotransferase, was severely reduced in strain SMS409. This indicated that the TnlO insertion in strain SMS409 inactivated aspC. An aspC mutant of E. coli was also inhibited by either sulfometuron methyl or tyrosine. We present a hypothesis which relates the observed a-ketobutyrate accumulation in sulfometuron methyl-inhibited cultures of strain SMS409 to aspartate starvation.

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“…While the two enzymes have a similar K m towards 2-ketoisovalerate, GDH2 has a five times higher k cat than GDHwt and can produce valine for cell growth in physiological condition. Compared to GDHwt, GDH2 has a significant enhanced catalytic activity on 2-ketobutyrate with a 4-fold decrease in K m (8.4 metabolic activities (24). On the basis of the crystallographic model for the active site of GDH, amino group of K92 hydrogenbonds to the γ-carboxyl group of 2-ketoglutarate and determines the substrate specificity (22).…”

Section: Resultsmentioning

confidence: 99%

Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine

Zhang

Cho

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

133

104

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The dramatic increase in healthcare cost has become a significant burden to the world. Many patients are denied the accessibility of medication because of the high price of drugs. Total biosynthesis of chiral drug intermediates is an environmentally friendly approach that helps provide more affordable pharmaceuticals. Here we have expanded the natural metabolic capability to biosynthesize a nonnatural amino acid L-homoalanine, which is a chiral precursor of levetiracetam, brivaracetam, and ethambutol. We developed a selection strategy and altered the substrate specificity of ammoniumassimilating enzyme glutamate dehydrogenase. The specificity constant k cat ∕K m of the best mutant towards 2-ketobutyrate is 50-fold higher than that towards the natural substrate 2-ketoglutarate. Compared to transaminase IlvE and NADH-dependent valine dehydrogenases, the evolved glutamate dehydrogenase increased the conversion yield of 2-ketobutyrate to L-homoalanine by over 300% in aerobic condition. As a result of overexpressing the mutant glutamate dehydrogenase and Bacillus subtilis threonine dehydratase in a modified threonine-hyperproducing Escherichia coli strain (ATCC98082, ΔrhtA), 5.4 g∕L L-homoalanine was produced from 30 g∕L glucose (0.18 g∕g glucose yield, 26% of the theoretical maximum). This work opens the possibility of total biosynthesis of other nonnatural chiral compounds that could be useful pharmaceutical intermediates. chiral chemicals | directed evolution | metabolic engineering | synthetic biology

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2-ketobutyrate: A putative alarmone of Escherichia coli

Cited by 32 publications

References 22 publications

A Mathematical Model for the Branched Chain Amino Acid Biosynthetic Pathways of Escherichia coli K12

A Mathematical Model for the Branched Chain Amino Acid Biosynthetic Pathways of Escherichia coli K12

Sensitivity of a Salmonella typhimurium aspC mutant to sulfometuron methyl, a potent inhibitor of acetolactate synthase II

Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine

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