Threonine-sensitive aspartokinase-homoserine dehydrogenase complex amino acid compositions, molecular weight, and subunit composition of the complex

Starnes, Willis L.; Munk, Petr; Maul, Stephen B.; Cunningham, Glenn N.; Cox, D. J.; Shive, William

doi:10.1021/bi00755a003

Cited by 37 publications

(13 citation statements)

References 29 publications

(18 reference statements)

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“…5A) to be enzymatically active, with specific activities of 5.6 and 6.8 U mg protein Ϫ1 , respectively (Table 1). These enzymatic activities are comparable to those of other biochemically characterized microbial aspartokinases (22,30,60). The kinetic data for the substrates ATP and L-aspartate of both Ask_LysC and Ask_Ect from P. stutzeri A1501 are summarized in Table 1.…”

Section: Purification Of the Ask_lysc And Ask_ect Proteinssupporting

confidence: 67%

A Specialized Aspartokinase Enhances the Biosynthesis of the Osmoprotectants Ectoine and Hydroxyectoine in Pseudomonas stutzeriA1501

et al. 2011

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The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. L-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/ hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. L-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from L-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an ask gene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_ask genes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. coli strain carrying the plasmid-based ectABCD_ask gene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCD gene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis.The growth arrest of microbial cells observed under severe high-osmolarity conditions is correlated with the reduction in the amount of free cytoplasmic water (12) and the reduction or collapse of turgor (7, 65). To promote water reentry and retention under high-osmolarity growth conditions and to balance turgor, many microorganisms amass a selected class of organic osmolytes, the compatible solutes (31, 66). Microbial cells accumulate compatible solutes through either synthesis or uptake from environmental resources (7, 65, 69). They can employ them not only as effective osmostress protectants, but also as protectants against heat (8, 10, 13, 17, 26) and cold (2, 25, 33) stress.The tetrahydropyrimidines ectoine and 5-hydroxyectoine ( Fig. 1) are an important class of compatible solutes (9,45,50). Biosynthesis of ectoine is catalyzed by L-2,4-diaminobutyric acid transaminase (EctB), N-␥-acetyltransferase (EctA), and ectoine synthase (EctC) (40,44). A subset of the ectoine producers hydroxylate ectoine to 5-hydroxyectoine (9, 17, 48) in a stereospecific fashion using the ectoine hydroxylase EctD (8, 9, 50). The structural genes (ectABC) for the ectoi...

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Section: Purification Of the Ask_lysc And Ask_ect Proteinssupporting

confidence: 67%

A Specialized Aspartokinase Enhances the Biosynthesis of the Osmoprotectants Ectoine and Hydroxyectoine in Pseudomonas stutzeriA1501

et al. 2011

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“…thrA and metL encode the fused aspartate kinase/homoserine dehydrogenase 1 and 2 (ASPK and HSDy), respectively. ASPK and HSDy catalyze the conversion of asp-L to L-aspartyl-4-phosphate (4pasp) and L-aspartate-semialdehyde (aspsa) to L-homoserine (hom-L) (Falcoz-Kelly et al, 1969 ; Starnes et al, 1972 ). metL gene expression is repressed by high met-L levels (Patte et al, 1967 ).…”

Section: Resultsmentioning

confidence: 99%

Growth Adaptation of gnd and sdhCB Escherichia coli Deletion Strains Diverges From a Similar Initial Perturbation of the Transcriptome

McCloskey

Sandberg

et al. 2018

Front. Microbiol.

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Adaptive laboratory evolution (ALE) has emerged as a new approach with which to pursue fundamental biological inquiries and, in particular, new insights into the systemic function of a gene product. Two E. coli knockout strains were constructed: one that blocked the Pentose Phosphate Pathway (gnd KO) and one that decoupled the TCA cycle from electron transport (sdhCDAB KO). Despite major perturbations in central metabolism, minimal growth rate changes were found in the two knockout strains. More surprisingly, many similarities were found in their initial transcriptomic states that could be traced to similarly perturbed metabolites despite the differences in the network location of the gene perturbations and concomitant re-routing of pathway fluxes around these perturbations. However, following ALE, distinct metabolomic and transcriptomic states were realized. These included divergent flux and gene expression profiles in the gnd and sdhCDAB KOs to overcome imbalances in NADPH production and nitrogen/sulfur assimilation, respectively, that were not obvious limitations of growth in the unevolved knockouts. Therefore, this work demonstrates that ALE provides a productive approach to reveal novel insights of gene function at a systems level that cannot be found by observing the fresh knockout alone.

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“…We chose AK, encoded by lysC 17, and ASD, encoded by asd 18, from E. coli , and HSD, encoded by HOM6 (ref. 19), from Saccharomyces cerevisiae since the HSD enzymes in E. coli are bifunctional enzymes with an associated AK activity2021. We found that Ec-Asd had only trace activity on the synthetic substrate ( k cat =0.13 s −1 on malyl-P versus 36 s −1 on aspartyl-4-phosphate (aspartyl-P), whereas Ec-LysC and Sc-Hom6 had no detectable activity on malate and malate semialdehyde, respectively (Supplementary Table 2; Supplementary Note 3).…”

Section: Resultsmentioning

confidence: 99%

Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid

et al. 2017

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2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l−1 DHB with a molar yield of 0.15.

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Threonine-sensitive aspartokinase-homoserine dehydrogenase complex amino acid compositions, molecular weight, and subunit composition of the complex

Cited by 37 publications

References 29 publications

A Specialized Aspartokinase Enhances the Biosynthesis of the Osmoprotectants Ectoine and Hydroxyectoine in Pseudomonas stutzeriA1501

A Specialized Aspartokinase Enhances the Biosynthesis of the Osmoprotectants Ectoine and Hydroxyectoine in Pseudomonas stutzeriA1501

Growth Adaptation of gnd and sdhCB Escherichia coli Deletion Strains Diverges From a Similar Initial Perturbation of the Transcriptome

Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid

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