Structure of the Regulatory Complex of
            <i>Escherichia coli</i>
            III
            <sup>Glc</sup>
            with Glycerol Kinase

Hurley, JH; Faber, H.R.; Worthylake, David K.; Meadow, N D; Roseman, Saul; Pettigrew, Donald W.; Remington, S.J.

doi:10.1126/science.8430315

Cited by 227 publications

(278 citation statements)

References 52 publications

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“…Sargent et al 10 have described a C256R substitution in a patient with 12% GK activity. This amino-acid residue is located in a b-sheet close to domains involved in ATP binding, 22 and this may result in an enzyme with some residual activity. The insertion of an Alu sequence in intron 4 of the GK gene described by Zhang et al 11 does not effect the mRNA as judged from their RT-PCR analysis, but the residual GK activity was 32% of the mean normal control.…”

Section: Discussionmentioning

confidence: 99%

“…Although the leucine 61 is only conserved in eukaryotes such as rat, mouse and Saccharomyces cerevisiae (Figure 5b), it is located in an a-helix on the outside of the GK subunit that interacts with another subunit to form the functional tetramer. 22 As proline residues are prone to interrupt helix formation, the mutation may cause a conformational change that influences the functioning of the GK protein.…”

Section: Discussionmentioning

confidence: 99%

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Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation

et al. 2004

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Four unrelated patients with glyceroluria ranging from 7 to 170 mmol/l were studied. The activity of glycerol kinase (GK) in cultured fibroblasts was determined with a specific enzyme assay and with two indirect methods, that is, incorporation into macromolecules of [ 14 C] from [ 14 C]glycerol and its oxidation to [14 C]CO 2 . Exon amplification and RT-PCR were used to identify mutations. In patient 1, with low activity in all three assays, we identified a c.1194A4C (E398D) missense mutation. In patient 2 with a considerable activity of the GK enzyme (22% of reference), oxidation to [ 14 C]CO 2 (37%) and a high incorporation of [ 14 C] into macromolecules (92%), we identified a c.182T4C (L61P) mutation that causes the enzyme to have a higher K m for glycerol (B300 lM) than normals (2 -8 lM). In patient 3, the GK activity estimated by the three different methods ranged from 16 to 22% of reference. Analysis of mRNA from the GK gene revealed three alternatively spliced transcripts. A mutation in intron 3 (g.16835G4A) resulted in an insertion of a cryptic exon between exon 2 or 3 and exon 4. Patient 4 with minor glyceroluria (7 mmol/l) and normal plasma glycerol concentration had normal activity with all three assay methods, thus excluding GK deficiency (GKD) as a cause of slight glyceroluria. To evaluate fully patients with glyceroluria, one needs to measure the GK activity and relate this and the clinical data to genetic findings. Residual enzyme activities in cultured fibroblasts can be found in GKD patients with severe clinical symptoms.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation

et al. 2004

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“…The unphosphorylated form of IIA Glc inhibits transport of non-PTS sugars such as lactose, maltose, melibiose, and raffinose by interacting with transporters for these sugars (a process termed inducer exclusion) (1,(2)(3)(4). Other allosteric regulatory functions of IIA Glc include inhibition of the phosphorylation of glycerol by binding to glycerol kinase (5) and either inhibition or activation of adenylyl cyclase (6).…”

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confidence: 99%

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

Koo

Yoon

Lee

et al. 2004

Journal of Biological Chemistry

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The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIA Glc (IIA Glc )) mediates some of these regulatory phenomena. In this report, we characterize a novel IIA Glc -binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as "function unknown." FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIA Glc , with the highest affinity of any protein thus far shown to interact with IIA Glc . Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the ␥-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIA Glc regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA. The bacterial phosphoenolpyruvate(PEP)1 :sugar phosphotransferase system (PTS) plays an important role in the transport of a variety of sugar substrates. This system catalyzes phosphorylation coupled to translocation of numerous simple sugars across the cytoplasmic membrane. The PTS is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein, HPr, which are used for all sugars, and, in addition, some sugar-specific components collectively known as enzymes II (1). The glucose-specific enzyme II of Escherichia coli consists of two components: soluble enzyme IIA Glc (IIA Glc ) and membrane-bound enzyme IICB Glc (IICB Glc ). Thus, glucose transport in E. coli involves three soluble PTS components (EI, HPr, and IIA Glc , encoded by the ptsHIcrr operon) and one membrane-bound protein, enzyme IICB Glc (encoded by the ptsG gene). Glucose uptake entails sequential phosphoryl transfer via the PTS, as follows: phosphoenolpyruvate (PEP) 3 EI 3 HPr 3 IIA Glc 3 IICB Glc 3 glucose. Components of the PTS also participate in several regulatory mechanisms (1). Catabolite repression allows for the preferential utilization of sugars transported by the PTS. Consequently, when E. coli are cultured in a medium containing both glucose and a non-PTS sugar, the glucose is consumed first. The currently accepted mechanism for this effect is that, when PTS sugars are transported, the steady-state condition of IIA Glc is mainly in the dephospho-form. The unphosphorylated form of IIA Glc inhibits transport of non-PTS sugars such as lactose, maltose, melibiose, and raffinose by interacting with transporters for these sugars (a process termed inducer exclusion) (1, 2-4). Other allosteric regulatory functions of IIA Glc incl...

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“…In addition to sugar transport, multiple roles are exerted by the PTS and these include chemoreception (3), catabolite repression (4), carbohydrate transport and metabolism (1,5,6), carbon storage (7,8), and the coordination of carbon and nitrogen metabolism (9). More recently, we found that EIIA Glc of the PTS also regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with the fermentation/respiration switch protein FrsA (10).…”

mentioning

confidence: 99%

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

Jeong

Kim

Cho

et al. 2004

Journal of Biological Chemistry

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Because the phosphoenolpyruvate:sugar phosphotransferase system plays multiple regulatory roles in addition to the phosphorylation-coupled transport of many sugars in bacteria, synthesis of its protein components is regulated in a highly sophisticated way. Thus far, the cAMP receptor protein (CRP) complex and Mlc are known to be the major regulators of ptsHIcrr and ptsG expression in response to the availability of carbon sources. In this report, we performed ligand fishing experiments by using the promoters of ptsHIcrr and ptsG as bait to find out new factors involved in the transcriptional regulation of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, and we found that the anaerobic regulator ArcA specifically binds to the promoters. Deletion of the arcA gene caused about a 2-fold increase in the ptsG expression, and overexpression of ArcA significantly decreased glucose consumption. In vitro transcription assays showed that phospho-ArcA (ArcA-P) represses ptsG P1 transcription. DNase I footprinting experiments revealed that ArcA-P binds to three sites upstream of the ptsG P1 promoter, two of which overlap the CRP-binding sites, and the ArcA-P binding decreases the CRP binding that is essential for the ptsG P1 transcription. These results suggest that the response regulator ArcA regulates expression of enzyme IICB Glc mediating the first step of glucose metabolism in response to the redox conditions of growth in E. coli.The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) 1 consists of two general cytoplasmic proteins, enzyme I and histidine phosphocarrier protein HPr, and some sugar-specific components collectively known as enzyme II (1, 2). Glucose-specific enzyme II of Escherichia coli consists of two subunits, soluble enzyme IIA Glc (EIIA Glc ) and membrane-bound enzyme IICB Glc . Thus, glucose transport in E. coli involves three soluble PTS components (enzyme I, HPr, and EIIA Glc , encoded by the ptsHIcrr operon) and one membranebound protein, enzyme IICB Glc (EIICB Glc ), encoded by the ptsG gene. During translocation of glucose, a phosphoryl group derived from phosphoenolpyruvate is transferred sequentially along a series of proteins to the transported glucose molecule, eventually converting it into glucose 6-phosphate. The sequence of phosphotransfer is from phosphoenolpyruvate to the general PTS proteins enzyme I and HPr and further to the carbohydrate-specific cytoplasmic EIIA Glc , membrane-bound EIICB Glc , and glucose.The PTS takes an important part in metabolic adaptation to environmental changes to compete effectively with ambient organisms by sensing the availability of nutrients in the environment. In addition to sugar transport, multiple roles are exerted by the PTS and these include chemoreception (3), catabolite repression (4), carbohydrate transport and metabolism (1, 5, 6), carbon storage (7,8), and the coordination of carbon and nitrogen metabolism (9). More recently, we found that EIIA Glc of the PTS also regulates the flux between respirat...

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Structure of the Regulatory Complex of Escherichia coli III ^Glc with Glycerol Kinase

Cited by 227 publications

References 52 publications

Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation

Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

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Structure of the Regulatory Complex of Escherichia coli III Glc with Glycerol Kinase

Cited by 227 publications

References 52 publications

Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation

Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

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Product

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Structure of the Regulatory Complex of Escherichia coli III ^Glc with Glycerol Kinase