Taking Control over Control: Use of Product Sensing in Single Cells to Remove Flux Control at Key Enzymes in Biosynthesis Pathways

Schendzielorz, Georg; Dippong, Martin; Grünberger, Alexander; Kohlheyer, Dietrich; Yoshida, Ayako; Binder, Stephan; Nishiyama, Chiharu; Nishiyama, Makoto; Bott, Michael; Eggeling, Lothar

doi:10.1021/sb400059y

Cited by 128 publications

(99 citation statements)

References 40 publications

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“…1 In addition to being a model for understanding allostery, 2-4 ATPPRT is of biotechnological interest as a tool for histidine production, provided that histidine feedback inhibition can be overcome. [5][6][7] Two forms of ATPPRT, encoded by the hisG gene, are found in nature. Fungi, plants, and most bacteria possess a long, homo-hexameric protein, HisGL, each subunit consisting of two domains that make up the catalytic core and a C-terminal regulatory domain that mediates feedback inhibition by histidine.…”

mentioning

confidence: 99%

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Stroek

Talbot²,

Glok³

et al. 2017

Biochemistry

104

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ATP-phosphoribosyltransferase (ATPPRT) catalyses the first step in histidine biosynthesis, the condensation of ATP and 5-phospho--D-ribosyl-1-pyrophosphate to generate N 1 -(5-phospho--D-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain, and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was solved at 2.34-Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-to 4-fold increase in kcat.Reaction profiles for both enzymes are indistinguishable by 31 P-NMR, indicating that the same reaction is catalysed. Temperature dependence of kcat shows deviation from Arrhenius behaviour at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 K and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterisation of a psychrophilic ATPPRT. 4Adenosine 5ʹ-triphosphate phosphoribosyltransferase (ATPPRT) (EC 2.4.2.17) catalyses the reversible Mg 2+ -dependent reaction between adenosine 5ʹ-triphosphate (ATP) and(PR-ATP) and inorganic pyrophosphate (PPi) (Scheme 1), the first step in histidine biosynthesis. 1 The chemical equilibrium of the reaction strongly favours reactants, 2 and the enzyme is allosterically inhibited by histidine. 1 In addition to being a model for understanding allostery, 2-4 ATPPRT is of biotechnological interest as a tool for histidine production, provided that histidine feedback inhibition can be overcome. [5][6][7] Two forms of ATPPRT, encoded by the hisG gene, are found in nature. Fungi, plants, and most bacteria possess a long, homo-hexameric protein, HisGL, each subunit consisting of two domains that make up the catalytic core and a C-terminal regulatory domain that mediates feedback inhibition by histidine. 8 Some bacteria and archaea have a short version of the protein, HisGS, which lacks the C-terminal regulatory domain and is insensitive to histidine. In these organisms, a catalytically inactive regulatory protein, HisZ, the product of the hisZ gene, is present. 9 HisZ, which shares a common ancestry with histidyl-tRNA synthetase (HisRS), binds HisGS to form ...

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mentioning

confidence: 99%

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Stroek

Talbot²,

Glok³

et al. 2017

Biochemistry

104

View full text Add to dashboard Cite

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“…In future applications, our device is going to be applied for analyzing extracellular and intracellular responses of single bacteria due to short term fluctuations in pH, temperature, carbon sources and others. This will be achieved, e.g., by the application of genetically encoded fluorescence sensors [3,4]. The layout can be applied to many other types of typically rod-shaped bacteria of similar size.…”

Section: Discussionmentioning

confidence: 99%

“…Microfluidic single-cell analysis assays can be divided into two main categories, namely studying either the whole cell (growth [1], morphology [2], or fluorescent bioreporters [3,4]) or its lysate (genome, transcriptome, proteome and metabolome) [5]. Emerging technologies such as genetically encoded bioreporters have extended the toolbox for noninvasive whole-cell single-cell analysis and have been applied to measure metabolic states such as intercellular pH [6] and product formation [3].…”

Section: Introductionmentioning

confidence: 99%

Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria

et al. 2013

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Abstract:Microfluidics has become an essential tool in single-cell analysis assays for gaining more accurate insights into cell behavior. Various microfluidics methods have been introduced facilitating single-cell analysis of a broad range of cell types. However, the study of prokaryotic cells such as Escherichia coli and others still faces the challenge of achieving proper single-cell immobilization simply due to their small size and often fast growth rates. Recently, new approaches were presented to investigate bacteria growing in monolayers and single-cell tracks under environmental control. This allows for high-resolution time-lapse observation of cell proliferation, cell morphology and fluorescence-coupled bioreporters. Inside microcolonies, interactions between nearby cells are likely and may cause interference during perturbation studies. In this paper, we present a microfluidic device containing hundred sub-micron sized trapping barrier structures for single E. coli cells. Descendant cells are rapidly washed away as well as components secreted by growing cells. Experiments show excellent growth rates, indicating high cell viability. Analyses of elongation and growth rates as well as morphology were successfully performed. This device will find application in prokaryotic single-cell studies under constant environment where by-product interference is undesired.

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“…2). As typical for long biosynthesis pathways, the aspartate kinase at the beginning of the pathway is feedback regulated, and a number of deregulated kinase enzymes can now be easily generated to result in increased Llysine formation (Chen et al 2011;Schendzielorz et al 2013). The first branch point is the distribution of aspartate semialdehyde towards either L-homoserine or L-lysine.…”

Section: Precursor Assembly Linementioning

confidence: 99%

“…In this manner, the rapid delivery of feedback resistant variants of aspartate kinase as w e l l a s N -a c e t y l -L -g l u t a m a t e k i n a s e a n d AT P phosphoribosyltransferase, key enzymes of L-arginine and Lhistidine synthesis, respectively, was achieved (Schendzielorz et al 2013). The realization of introducing chromosomal mutations in C. glutamicum by just using oligonucleotides (recombineering) offers even more exiting possibilities (Fig.…”

Section: New Screening Techniquesmentioning

confidence: 99%

A giant market and a powerful metabolism: l-lysine provided by Corynebacterium glutamicum

Eggeling

Bott

2015

Appl Microbiol Biotechnol

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196

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L-lysine is made in an exceptional large quantity of currently 2,200,000 tons/year and belongs therefore to one of the leading biotechnological products. Production is done almost exclusively with mutants of Corynebacterium glutamicum. The increasing L-lysine market forces companies to improve the production process fostering also a deeper understanding of the microbial physiology of C. glutamicum. Current major challenges are the identification of ancillary mutations not intuitively related with product increase. This review gives insights on how cellular characteristics enable to push the carbon flux in metabolism towards its theoretical maximum, and this example may also serve as a guide to achieve and increase the formation of other products of interest in microbial biotechnology.

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Taking Control over Control: Use of Product Sensing in Single Cells to Remove Flux Control at Key Enzymes in Biosynthesis Pathways

Cited by 128 publications

References 40 publications

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria

A giant market and a powerful metabolism: l-lysine provided by Corynebacterium glutamicum

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