Escherichia coli Glycerol Kinase: Role of a Tetramer Interface in Regulation by Fructose 1,6-Bisphosphate and Phosphotransferase System Regulatory Protein IIIglc

Liu, Wei Zhang; Faber, Rachel M.; Feese, M.D.; Remington, S.J.; Pettigrew, Donald W.

doi:10.1021/bi00199a040

Cited by 42 publications

(68 citation statements)

References 23 publications

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“…Surprisingly, two of the alternate alleles (A218T and G217T) affected the same amino acid (Asp73) as the A218T mutation in clone GB-1. A mutation in this amino acid was identified in a previous study 25 and was shown to decrease inhibition by FBP and the formation of inactive tetramers. For the A218T mutation in GD, we were able to rule out cross-contamination because the accompanying rpoC mutation from GB was not present in GD colonies.…”

Section: Mutation Frequencies In Evolving Populationsmentioning

confidence: 88%

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Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale

et al. 2006

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We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.Comparative genomics has been almost entirely focused on genomic changes over long periods of time, on the order of millions of years. A new microarray-based method of whole-genome resequencing called comparative genome sequencing (CGS) 1 now makes it cost efficient to monitor bacterial evolution comprehensively over short time periods as well. This capability is important because many microbial phenomena, such as the emergence of new pathogens and the acquisition of antibiotic resistance factors, can occur over relatively short time scales. Experimental evolution of bacteria and viruses 2,3 is a facile approach to study these topics. It has been used to test predictions of evolutionary theory 4 and to study parallel changes in populations evolved for 20,000 generations 5 , acquisition of antibiotic resistance 1 and in vitro symbiosis 6 . Our laboratory has used experimental evolution as a tool for metabolic engineering 7 and to study the recovery of strains with gene knockouts in central metabolic genes 8 . Nevertheless, much remains unknown about genome plasticity over short evolutionary timescales.It is a common mistake to think of bacteria as static; that is, to assume that a culture grown overnight is the same as it was the day before. It has been estimated that nearly 10% of the individual bacteria in a Salmonella enterica population carry large-scale genome rearrangements 9 , and in a suboptimal environment, selection can alter a population very rapidly. The 10-20 generations that occur in the process of growing a bacterial culture are sufficient to create a heterogeneous population, depending on the magnitude of the selective advantage of adaptive mutations. This problem is avoided by using strains of bacteria that are adapted to common laboratory media, but there are interesting cases where a seemingly straightforward growth medium poses a great challenge to a bacterium.An example is E. coli K-12 grown in minimal medium supplemented with glycerol as the carbon and energy source. Despite a complete pathway for glycerol catabolism, large variations in the growth rates of various strains have been noted 10 . Growth of the sequenced strain MG1655 has been observed to differ from computational predictions based on flux balance analysis 11 . Upon extended logarithmic growth in glycerol minimal medium, the growth rate...

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Section: Mutation Frequencies In Evolving Populationsmentioning

confidence: 88%

“…Every day, optical density (OD) measurements were made and cells were diluted into fresh medium, estimating the amount of inoculum to use such that the culture would not enter stationary phase. Samples were frozen at days 2,4,6,8,10,15,20,25,30,35, 40 and 44.…”

Section: Methodsmentioning

confidence: 99%

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale

et al. 2006

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“…Escherichia coli strain MV1190 or DG1 [37] was transformed with an expression vector, pT3-5LO, which contains a human 5LO cDNA insert [38]. An overnight culture was grown at 30 mC in Luria-Bertani medium containing 150 µg\ml ampicillin.…”

Section: Preparation Of 5lomentioning

confidence: 99%

Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences

Hammarberg

Rådmark

Samuelsson

et al. 2000

Biochemical Journal

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5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP ; however, a consensus ATP-binding site or nucleotidebinding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5h-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA\mol of 5LO (of which ATP competed with 1 mol\mol) or 0.94 mol of 2-azido-ATP\mol of 5LO (of which ATP competed with 0.77 mol\mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site

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“…1C). In solution, EcGK shows an equilibrium between homodimers and homotetramers (18), and FBP inhibition requires tetramer formation (19). Sedimentation velocity ultracentrifugation shows that HiGK sediments more slowly than EcGK in the absence of FBP, suggesting the presence of a greater fraction of dimer but about the same as EcGK in the presence of FBP.…”

mentioning

confidence: 97%

“…Allosteric inhibition of EcGK by FBP and IIA Glc operate independently, and IIA Glc inhibition does not require tetramer formation (19)(20)(21). Thus, HiGK is naive with respect to allosteric control by E. coli IIA Glc .…”

mentioning

confidence: 99%

Transplanting allosteric control of enzyme activity by protein–protein interactions: Coupling a regulatory site to the conserved catalytic core

Pawlyk

Pettigrew²

2002

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

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Glycerol kinase from Escherichia coli, but not Haemophilus influenzae, is inhibited allosterically by phosphotransferase system protein IIA Glc . The primary structures of these related kinases contain 501 amino acids, differing at 117. IIA Glc inhibition is transplanted from E. coli glycerol kinase into H. influenzae glycerol kinase by interconverting only 11 of the differences: 8 residues that interact with IIA Glc at the allosteric binding site and 3 residues in the conserved ATPase catalytic core that do not interact with IIA Glc but the solvent accessible surface of which decreases when it binds. The three core residues are crucial for coupling the allosteric site to the conserved catalytic core of the enzyme. The site of the coupling residues identifies a regulatory locus in the sugar kinase͞ heat shock protein 70͞actin superfamily and suggests relations between allosteric regulation and the active site closure that characterizes the family. The location of the coupling residues provides empirical validation of a computational model that predicts a coupling pathway between the IIA Glc A llosteric regulation of enzyme catalysis is a fundamental aspect of control in biological systems. For most classically studied allosteric enzymes (1), catalytic activity is modulated by small molecules such as fructose 1,6-bisphosphate (FBP), phosphoenolpyruvate, or nucleotides. The regulatory binding sites and the active sites are located at subunit-subunit interfaces of the homooligomeric proteins and are separated by Ͼ10 Å. Substrate binding shows homotropic interactions, typically positive cooperativity. Allosteric effectors change the affinity for substrates at the active sites but do not affect V max . Allosteric inhibition decreases apparent affinity for substrate, and activation increases apparent affinity. Changes in quaternary structure, involving small rigid-body rotations and translations of subunits, are seen in crystal structures of the enzymes with substrates or allosteric effectors. Regulation of catalysis by protein-protein interactions is less understood but seems to be the rule rather than the exception, and there is widespread interest in interactomes and roles of macromolecular interactions in living systems (2). A related area of interest is the molecular evolution of allosteric control by protein-protein interactions. Here we show that transplantation of allosteric regulation from one enzyme to another provides insights into how protein-protein interactions distant from an active site regulate catalysis and into mechanisms of molecular evolution.Glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) from Escherichia coli (EcGK) is a regulatory enzyme (3). It is inhibited allosterically by IIA Glc , the 18-kDa glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (4). Binding of IIA Glc decreases V max and the substrate Michaelis constants, and no cooperativity is observed for inhibition or with respect to glycerol or MgATP (5).Thus, in contra...

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Escherichia coli Glycerol Kinase: Role of a Tetramer Interface in Regulation by Fructose 1,6-Bisphosphate and Phosphotransferase System Regulatory Protein IIIglc

Cited by 42 publications

References 23 publications

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale

Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences

Transplanting allosteric control of enzyme activity by protein–protein interactions: Coupling a regulatory site to the conserved catalytic core

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