FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1997495869> ?p ?o ?g. }

• W1997495869 endingPage "12238" @default.
• W1997495869 startingPage "12234" @default.
• W1997495869 abstract "The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IICMan-IIDMan complex and the cytoplasmic IIABMan subunit. IIABMan has two flexibly linked domains, IIAMan and IIBMan, each containing a phosphorylation site (His-10 and His-175). Phosphoryl groups are transferred from the phosphoryl carrier protein phospho-HPr to His-10, hence to His-175 and finally to the 6′ OH of the transported hexose. Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions. IIBMan contains five arginines which are invariant in the homologous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis. The IIA domains have no conserved arginines. The five arginines were replaced by Lys or Gln one at a time, and the mutants were analyzed for transport and phosphorylation activity. All five IIB mutants can still be phosphorylated at His-175 by the IIA domain. R172Q is completely inactive with respect to glucose phosphotransferase (phosphoryltransfer from His-175 to the 6′ OH of Glc) and hexose transport activity. R168Q has no hexose transport and strongly reduced phosphotransferase activity. R204K has no transport but almost normal phosphotransferase activity. R304Q has only slightly reduced transport activity. R190K behaves like wild-type IIABMan. Arg-168, Arg-172, and Arg-304 are part of the hydrogen bonding network on the surface of IIB, which contains the active site His-175 and the interface with the IIA domain (Schauder, S., Nunn, R.S., Lanz, R., Erni, B. and Schirmer, T. (1998) J. Mol. Biol. 276, 591–602) (Protein Data Bank accession code 1BLE). Arg-204 is at the putative interface between IIBMan and the IICMan-IIDMan complex. The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IICMan-IIDMan complex and the cytoplasmic IIABMan subunit. IIABMan has two flexibly linked domains, IIAMan and IIBMan, each containing a phosphorylation site (His-10 and His-175). Phosphoryl groups are transferred from the phosphoryl carrier protein phospho-HPr to His-10, hence to His-175 and finally to the 6′ OH of the transported hexose. Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions. IIBMan contains five arginines which are invariant in the homologous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis. The IIA domains have no conserved arginines. The five arginines were replaced by Lys or Gln one at a time, and the mutants were analyzed for transport and phosphorylation activity. All five IIB mutants can still be phosphorylated at His-175 by the IIA domain. R172Q is completely inactive with respect to glucose phosphotransferase (phosphoryltransfer from His-175 to the 6′ OH of Glc) and hexose transport activity. R168Q has no hexose transport and strongly reduced phosphotransferase activity. R204K has no transport but almost normal phosphotransferase activity. R304Q has only slightly reduced transport activity. R190K behaves like wild-type IIABMan. Arg-168, Arg-172, and Arg-304 are part of the hydrogen bonding network on the surface of IIB, which contains the active site His-175 and the interface with the IIA domain (Schauder, S., Nunn, R.S., Lanz, R., Erni, B. and Schirmer, T. (1998) J. Mol. Biol. 276, 591–602) (Protein Data Bank accession code 1BLE). Arg-204 is at the putative interface between IIBMan and the IICMan-IIDMan complex. Protein phosphorylation plays an important role in energy transduction, signal transduction and enzyme regulation. P-type ATPases transport cations across the cell membrane and are transiently phosphorylated on an aspartic acid during turnover (1Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (416) Google Scholar). Protein kinases and protein phosphatases regulate the activity of enzymes and membrane bound receptors by phosphorylation and dephosphorylation (2Bourret R.B. Borkowich K.A. Simon M.I. Annu. Rev. Biochem. 1991; 60: 401-441Crossref PubMed Scopus (390) Google Scholar, 3Fantl W.J. Johnson D.E. Williams L.T. Annu. Rev. Biochem. 1993; 62: 453-481Crossref PubMed Scopus (929) Google Scholar). In the phosphoenolpyruvate-dependent phosphotransferase system of bacteria (PTS) 1The abbreviations used are: PTS, phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (EC 2.7.1.69); IIABMan, hydrophilic subunit of the mannose transporter; IICMan and IIDMan, transmembrane subunits of the mannose transporter; IICBGlc, transmembrane subunit of the glucose transporter of the PTS; HPr, histidine-containing phosphocarrier protein of the PTS; PEP, phosphoenolpyruvate.1The abbreviations used are: PTS, phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (EC 2.7.1.69); IIABMan, hydrophilic subunit of the mannose transporter; IICMan and IIDMan, transmembrane subunits of the mannose transporter; IICBGlc, transmembrane subunit of the glucose transporter of the PTS; HPr, histidine-containing phosphocarrier protein of the PTS; PEP, phosphoenolpyruvate. active transport and signaling are two functions of a protein phosphorylation cascade comprising four phosphoprotein units. The four components, enzyme I, HPr, IIA, and IIB sequentially transfer phosphoryl groups from phosphoenolpyruvate to carbohydrates that are accumulated across the cell membrane by a mechanism coupling translocation to phosphorylation. Whereas enzyme I and HPr are two energy coupling components, IIA and IIB together with the transmembrane IIC (and sometimes an additional IID) units form the sugar-specific transport complexes. IIA, IIB, and IIC occur either as protein subunits or as domains of a multidomain protein. Escherichia coli for example has over 30 genes for transporter complexes that differ in substrate specificity, amino acid sequence, and subunit/domain composition. The phosphorylation sites are histidines in enzyme I, HPr and the different IIA components, cysteines in the IIBs belonging to the glucose, mannitol, and lactose family, and histidines in the IIBs belonging to the mannose family of PTS transporters (for reviews see Refs. 4Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar and 5Postma P.W. Lengeler J.W. Jacobson G.R. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella, Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 1149-1174Google Scholar). Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions (6Johnson L.N. Barford D. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 199-232Crossref PubMed Scopus (219) Google Scholar). Amino acid sequence comparisons with respect to invariant arginines in PTS proteins from different bacteria reveal the following picture. The N-terminal domain of enzyme I has Arg-126, Arg-186, and Arg-195, which are completely invariant in 17 sequences. Arg-186 and Arg-195 are close to the phosphorylation site His-189 (7Liao D.I. Silverton E. Seok Y.J. Lee B.R. Peterkofsky A. Davies D.R. Structure (Lond.). 1996; 4: 861-872Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Arg-131 is conserved in 14 and Arg-136 is conserved in 15 sequences. The C-terminal domain has 7 completely invariant arginines (Protein Domain Data Base:protein.toulouse.inra.fr/prodom/prodom.html). The functional role of these residues in enzyme I has not been explored. HPr is a 9-kDa open faced four-stranded antiparallel β-sheet with three α-helices on one face. Arg-17, which is close to the active site His-15, is invariant in all 22 known sequences. When Arg-17 is replaced by Ser or Glu, phospho-donor and -acceptor activity of HPr are reduced to between 6% and less than 0.1% of the control (8Anderson J.W. Pullen K. Georges F. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 12325-12333Abstract Full Text PDF PubMed Google Scholar). The phosphoryl group bound to His-15 of HPr is most likely complexed by the guanidino group of Arg-17, as inferred from its restricted conformational freedom observed upon phosphorylation of HPr, and from 31P chemical shift changes (9Rajagopal P. Waygood E.B. Klevit R.E. Biochemistry. 1994; 33: 15271-15282Crossref PubMed Scopus (48) Google Scholar). However, more recently this stabilization of the phosphoryl group by the guanidino group has been questioned, based on a molecular dynamics simulation in water and refined NMR data of phospho-HPr (10Van Nuland N.A.J. Boelens R. Scheek R.M. Robillard G.T. J. Mol. Biol. 1995; 246: 180-193Crossref PubMed Scopus (80) Google Scholar, 11Jones B.E. Rajagopal P. Klevit R.E. Protein Sci. 1997; 10: 2107-2119Google Scholar). No invariant arginines occur in the 21 IIA components of the glucose family and in the five IIA domains of the mannose family of PTS transporters. Two arginines, Arg-424 and Arg-426, are invariant in the IIB components of the glucose family. They are close to the active site Cys-421 (residues numbers refer to theE. coli IICBGlc subunit), and both are essential for phospho-donor activity toward glucose but not for phospho-acceptor activity (12Lanz R. Erni B. J. Biol. Chem. 1998; 273: 12239-12243Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Five arginine residues, Arg-168, Arg-172, Arg-190, Arg-204, and Arg-304 (numbers refer to E. coliIIABMan subunit), are invariant in the IIB domains of the PTS transporters of E. coli, Klebsiella pneumoniae (13Wehmeier U.F. Lengeler J.W. Biochim. Biophys. Acta. 1994; 1208: 348-351Crossref PubMed Scopus (36) Google Scholar), and Bacillus subtilis (14Martin Verstraete I. Debarbouille M. Klier A. Rapoport G. J. Mol. Biol. 1990; 214: 657-671Crossref PubMed Scopus (114) Google Scholar) belonging to the mannose family. Arg-168 and Arg-172 are close in sequence to the active site His-175 of IIABMan. In summary, it appears that arginines close to the active site residues occur in the even numbered phosphorylation sites (HPr and IIB) of the glucose and mannose specific PTS and possibly in enyzme I but that arginines are absent from the IIA domains. The mannose transporter complex of E. coli consists of three subunits. The IICMan and IIDMan subunits comprise six and one putative membrane-spanning segments (15Huber F. Erni B. Eur. J. Biochem. 1996; 239: 810-817Crossref PubMed Scopus (37) Google Scholar). They contain the substrate binding site, and they facilitate the penetration of bacteriophage λ DNA by an as yet unknown mechanism (16Elliott J. Arber W. Mol. Gen. Genet. 1978; 161: 1-8Crossref PubMed Scopus (58) Google Scholar, 17Erni B. Zanolari B. Kocher H.P. J. Biol. Chem. 1987; 262: 5238-5247Abstract Full Text PDF PubMed Google Scholar). Two IIABMan subunits form a stable homodimer, which reversibly binds to the IICMan-IIDMan complex (K D 5–10 nm). The IIABMansubunit consists of two protein domains (IIA and IIB), which are connected by a 20-residue-long hinge peptide rich in alanine and proline (18Erni B. Zanolari B. Graff P. Kocher H.P. J. Biol. Chem. 1989; 264: 18733-18741Abstract Full Text PDF PubMed Google Scholar). The two domains can be separated by limited trypsinolysis at two lysine residues in the hinge. The IIAMan domain is phosphorylated by phospho-HPr at His-10 and donates the phosphoryl group to His-175 of the IIBMan domain. IIBManthen donates the phosphate to the sugar. Although IIBMancan accept a phosphate from IIAMan in the absence of the IICMan-IIDMan complex, it can donate the phosphate to the sugar only in the ternary complex with IICMan and IIDMan. The IIAMandomain has an α/β open pleated sheet structure. The central β-sheet consists of four parallel strands from one subunit and one antiparallel strand from the second subunit in the dimer (19Nunn R.S. Markovic-Housley Z. Génovésio-Taverne J.C. Flükiger K. Rizkallah P.J. Jansonius J.N. Schirmer T. Erni B. J. Mol. Biol. 1996; 259: 502-511Crossref PubMed Scopus (61) Google Scholar). This β strand exchange makes the dimer highly resistant to subunit dissociation. While this work was in progress, the x-ray structure of IIBLev of B. subtilis was solved (20Schauder S. Nunn R.S. Lanz R. Erni B. Schirmer T. J. Mol. Biol. 1998; 276: 591-602Crossref PubMed Scopus (38) Google Scholar). It has an open pleated α/β fold consisting of a strongly twisted seven-stranded β-sheet with helices on both faces (Fig. 4). The IIBMan domain has 47% amino acid sequence identity with IIBLev, and the two proteins complement each other (20Schauder S. Nunn R.S. Lanz R. Erni B. Schirmer T. J. Mol. Biol. 1998; 276: 591-602Crossref PubMed Scopus (38) Google Scholar). Here we characterize the functions of the invariant arginines in the IIBMan domain of the mannose transporter. E. coliK12-UT580ΔLPM (ΔmanXYZ::cat) and ZSC112LΔLPM(ΔmanXYZ::cat ptsG glk) carry a deletion of the chromosomal manXYZ allele. Gene replacement was done in E. coli DPE271 (recD) as described (21Buhr A. Flükiger K. Erni B. J. Biol. Chem. 1994; 269: 23437-23443Abstract Full Text PDF PubMed Google Scholar) with the following modifications. (i) TheHindIII-KpnI fragment encoding residues 111–323 of IIABMan, the complete IICMan, and residues 1–241 of IIDMan was removed from plasmid pTSML1 (22Erni B. Zanolari B. J. Biol. Chem. 1985; 260: 15495-15503Abstract Full Text PDF PubMed Google Scholar) and replaced by KpnI-HindIII fragment from plasmid pMc5–8 (23Stanssens P. Opsomer C. McKeown Y.M. Kramer W. Zabeau M. Fritz H.J. Nucleic Acids Res. 1989; 17: 4441-4445Crossref PubMed Scopus (260) Google Scholar) encoding cat (the KpnI site was introduced into the original pMc5–8 by gapped duplex site-directed mutagenesis). (ii) The recombinant plasmid pTSΔLPM::Cm was linearized with NdeI and used to transform E. coli DPE271 (recD). (iii) Transformants defective in mannose uptake were selected on McConkey indicator plates containing mannose and chloramphenicol and tested for the loss of ampicillin resistance. (iv) The cat gene was then P1 transduced into UT580 and ZSC112L. ZSC112ΔLPM was transformed with plasmid pTSPM6 (encoding IICMan and IIDMan) (18Erni B. Zanolari B. Graff P. Kocher H.P. J. Biol. Chem. 1989; 264: 18733-18741Abstract Full Text PDF PubMed Google Scholar) and a second plasmid encoding the IIABMan mutants, and the strains were used for in vivo experiments. WA2127ΔHIC (manXYZ, ΔptsH ptsI crr) (24Mao Q. Schunk T. Flükiger K. Erni B. J. Biol. Chem. 1995; 270: 5258-5265Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) was used as host for protein expression. Cells were grown at 37 °C in LB medium containing appropriate antibiotics. XL1-blue (Stratagene) was used for cloning and plasmid amplification. IIABMan mutants R168Q,R172Q, R304Q were constructed using the gapped duplex procedure (23Stanssens P. Opsomer C. McKeown Y.M. Kramer W. Zabeau M. Fritz H.J. Nucleic Acids Res. 1989; 17: 4441-4445Crossref PubMed Scopus (260) Google Scholar, 25Stolz B. Huber M. Markovic-Housley Z. Erni B. J. Biol. Chem. 1993; 268: 27094-27099Abstract Full Text PDF PubMed Google Scholar). Mutant clones were identified by way of diagnostic restriction sites FspI, PvuII and SacI respectively. Mutants R190K and R204K were constructed by overlap extension mutagenesis (26Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6810) Google Scholar). Mutant clones were identified by DNA sequencing. The HindIII-SnaBI segments carrying the mutations were cloned into the expression vector pJFL (encodingmanX under the control of tacP, identical with pTACL293) (27Seip S. Balbach J. Behrens S. Kessler H. Flükiger K. Demeyer R. Erni B. Biochemistry. 1994; 33: 7174-7183Crossref PubMed Scopus (22) Google Scholar). E. coli WA2127ΔHIC was transformed with derivatives of pJFL encoding wild-type and mutant IIABMan. IIABManwas overexpressed and purified as described (25Stolz B. Huber M. Markovic-Housley Z. Erni B. J. Biol. Chem. 1993; 268: 27094-27099Abstract Full Text PDF PubMed Google Scholar). Enzyme I and HPr were purified as described (24Mao Q. Schunk T. Flükiger K. Erni B. J. Biol. Chem. 1995; 270: 5258-5265Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The IICMan-IIDMancomplex was purified by metal chelate affinity chromatography (28Huber, F., Topology, Purification, and Subunit Composition of the Mannose Transporter of Escherichia coli.Ph.D. thesis, 1996, University of Bern.Google Scholar). 2R. Gutknecht, R. Lanz, and B. Erni, unpublished results. In vitrophosphorylation of [14C]Glc was assayed by ion-exchange chromatography as described (25Stolz B. Huber M. Markovic-Housley Z. Erni B. J. Biol. Chem. 1993; 268: 27094-27099Abstract Full Text PDF PubMed Google Scholar). 100 μl of incubation mixture contained 0.5 μg of enzyme I, 2.8 μg of HPr, and 0.5 μl of crude membranes (∼4 μg protein) containing the IICMan-IIDMan complex. Uptake of [14C]dGlc by bacteria was assayed as described (29Buhr A. Daniels G.A. Erni B. J. Biol. Chem. 1992; 267: 3847-3851Abstract Full Text PDF PubMed Google Scholar). The [14C]dGlc concentration was 120 μm, the specific activity was 6600 dpm/nmol. The cell density in the assay mixture wasA 600 = 15. 100-μl aliquots were removed at the indicated time points, diluted in ice-cold minimal salts medium, and applied to glass fiber filters under suction. The rate and the extent of protein phosphorylation and dephosphorylation was measured in a filter binding assay (21Buhr A. Flükiger K. Erni B. J. Biol. Chem. 1994; 269: 23437-23443Abstract Full Text PDF PubMed Google Scholar). The incubation mixture (50 mm NaPi, pH 7.4, 5 mmMgCl2, 2.5 mm NaF, 2.5 mmdithiothreitol) contained 15 μg of enzyme I, 2.5 μg of HPr, and 85 μg of IIABMan per 250 μl. The phosporylation reaction was started by adding to the incubation mixture at 24 °C [32P]PEP (∼1200 dpm/nmol) to a final concentration of 80 μm. Aliquots of 40 μl were withdrawn at the indicated time points and diluted into 1 ml of 80% ammonium sulfate solution at 4 °C. The protein precipitates were collected on glass microfiber filters (GF/F, Whatman) under suction. The precipitate was washed with 2× 1 ml of ice-cold 80% ammonium sulfate, and the filters were counted in a liquid scintillation counter. To measure the dephosphorylation rate of PIIABMan (rate of P transfer to mannose) the reaction mixture was first incubated with 80 μm [32P]PEP for 5 min (pulse). At time 0 the following components were added together: a 50-fold molar excess of unlabeled PEP (chase); 1 μg of IICMan/IIDMan; an aqueous suspension of E. coli lipids (Sigma) to a final concentration of 0.5 mg/ml; Glc to a final concentration of 0.4 mm. 40-μl aliquots were withdrawn and processed as indicated above. Control reactions containing all components with the exception of IICMan/IIDMan were done in parallel. The background counts due to enzyme I and HPr (less than 10%) were subtracted from the counts of the complete system. In parallel, the phosphorylated proteins were analyzed on 17.5% polyacrylamide gels as described (21Buhr A. Flükiger K. Erni B. J. Biol. Chem. 1994; 269: 23437-23443Abstract Full Text PDF PubMed Google Scholar). 20-μl incubation mixtures contained 100 μm [32P]PEP (<1200 dpm/nmol), 1 μg of enzyme I, 1 μg of HPr, 6 μg of IIABMan, and where indicated 2 μg of IICMan/IIDMan. [32P]PEP was prepared as described (30Roossien F.F. Brink J. Robillard G.T. Biochim. Biophys. Acta. 1983; 760: 185-187Crossref PubMed Scopus (59) Google Scholar). Expression of the three subunits of the mannose transporter was directed by two compatible plasmids encoding the soluble IIABMan subunit and the transmembrane IICMan-IIDMan complex. The expression of IICMan/IIDMan was constitutive. Expression of IIABMan was induced with 30 μmisopropyl-1-thio-β-d-galactopyranoside, at which concentration the cell growth rate on a mineral salts medium containing mannose as the only carbon source was maximal.2 Cells expressing wild-type IIABMan or mutants R190K and R304Q produced red colonies on McConkey mannose indicator plates, whereas mutants R168Q and R172Q produced yellow colonies. R204K produced yellow colonies with a small red center. The initial rates of [14C]dGlc uptake by wild-type and R190K expressing cells were very similar, whereas the R304Q mutant had about 50% of wild-type activity (Fig. 1 A). The arginine mutants R168Q, R172Q and R204K had less than 5% transport activity. The results from McConkey plates and in vivotransport studies are consistent and indicate that arginines 168, 172, and 204 are essential for transport activity of the mannose transporter, whereas arginines 190 and 304 are dispensable when replaced by lysine and glutamine, respectively. As expected, these conservative replacements did not have a major impact on structural stability and folding since all five mutant proteins could be overexpressed and also retained the strong affinity for phosphocellulose characteristic of IIABMan (not shown). They differ from the H175C active site mutant, which is no longer phosphorylated and displays a reduced affinity for phosphocellulose (25Stolz B. Huber M. Markovic-Housley Z. Erni B. J. Biol. Chem. 1993; 268: 27094-27099Abstract Full Text PDF PubMed Google Scholar). In the intact cell the PTS transporters catalyze vectorial transport coupled to phosphorylation of the substrate. Observations made with the glucose transporter indicate that phosphorylation of intracellular substrates by the PTS (nonvectorial phosphorylation) also occurs in intact cells. The physiological significance of this activity is unknown (31Thompson J. Chassy B.M. J. Bacteriol. 1985; 162: 224-234Crossref PubMed Google Scholar, 32Nuoffer C. Zanolari B. Erni B. J. Biol. Chem. 1988; 263: 6647-6655Abstract Full Text PDF PubMed Google Scholar). Phosphorylation without transport can be assayed with purified proteins. Purified IIABMan proteins were incubated in the presence of PEP and [14C]Glc, enzyme I, HPr, and crude membrane vesicles containing the IICMan-IIDMancomplex, and the formation of [14C]Glc6P was measured (Fig. 1 B). The R190K and R304Q mutants had the same activity as wild-type IIABMan, R204K had 70% and R168Q had 10% activity. The R172Q mutant was completely inactive. These in vitro results are consistent with the observations made with intact cells with one exception; the R204K mutant, which had a very lowin vivo transport activity, retained 70% nonvectorial phosphorylation activity. The IIABMan subunit consists of two independent domains (IIA and IIB) each containing one phosphorylation site. IIB accepts the phosphoryl groups from the IIAMan domain. All five invariant arginines are located in the IIB domain. How does the substitution of the invariant arginines affect phosphorylation at the two active sites? The arginine mutants of IIAB were phosphorylated with [32P]PEP in the presence of enzyme I and HPr. Thereafter the IIAMan and IIBMan domains were cleaved by limited trypsinolysis and separated by gel electrophoresis. All mutant proteins were phosphorylated on IIAMan (as expected) and also on the mutated IIBMan domains (Fig.2 A). The amount of32P in the IIB band is lower than in the IIA band because histidine Nδ phosphates are more sensitive to hydrolysis than histidine Nε phosphates and because the IIB domain also is more sensitive to incipient trypsinolysis than IIA (18Erni B. Zanolari B. Graff P. Kocher H.P. J. Biol. Chem. 1989; 264: 18733-18741Abstract Full Text PDF PubMed Google Scholar). The ratio32P-IIB to 32P-IIA after gel electrophoresis is between 0.3 and 0.4 for wild-type, R190K, and R304Q and between 0.13 and 0.15 for the R168Q, R172Q and R204K mutants. This suggests that the less active mutants are phosphorylated less efficiently or are less stable toward hydrolysis during gel electrophoresis. The addition of the IICMan-IIDMan complex and glucose in excess of [32P]PEP results in the complete dephosphorylation of all the phosphorylated proteins with the only exception of the R172Q mutant, indicating that the R172Q mutant can accept a phosphoryl group but is unable to donate it to glucose (Fig.2 B, + lanes). When glucose is omitted but the IICMan-IIDMan complex is present, wild-type IIABMan and the fully active R190K and R304Q are phosphorylated to a lesser degree than the less active R168Q, R172Q and R204K mutants (Fig. 2 B, − lanes). This is possibly due to IICMan/IIDMan-mediated spontaneous hydrolysis, which is faster with active IIABManthan with the less active IIABMan mutants. For a more quantitative analysis, the time course of phosphorylation and dephosphorylation was determined in solution. Purified IIABMan was incubated with enzyme I, HPr, and [32P]PEP, and aliquots were removed at the indicated time points (Fig. 3 A). R168Q,R172Q,R190K are phosphorylated at 50–75% the rate of the wild-type in the presence of a saturating concentration of HPr. The remaining mutants are phosphorylated at more than 80% the rate. The small difference vanishes when the HPr concentration is rate-limiting (Fig. 3 B). The dephosphorylation rates were measured in a pulse-chase experiment. IIABMan was labeled with32P in the presence of enzyme I and HPr. The chase was triggered by the addition of an excess of IICMan-IIDMan complex, glucose as P-acceptor and unlabeled PEP. The dephosphorylation rate is maximal for wild-type IIABMan. R204K, R190K, and R304Q dephosphorylate at an intermediate rate whereas R168Q and R172Q are very slow (Fig. 3C). A difference between R168Q and R172Q could be seen when the IICMan/IIDMan concentration was increased. The rate of dephosphorylation of R168Q as well as of wild-type IIABMan increased with increasing IICMan/IIDMan concentration, whereas R172Q remained inactive irrespective of the IICMan/IIDMan concentration (Fig.3 D).Figure 3Time course of phosphorylation and dephosphorylation of IIABMan. A, purified IIABMan was incubated with [32P]PEP in the presence of catalytic amounts of enzyme I and HPr. The reaction was stopped at the indicated time points by ammonium sulfate precipitation. Protein precipitates were collected on filters and counted as described under “Experimental Procedures.” B, same as Abut in the presence of a rate-limiting concentration (90 μg/ml) of HPr. C, purified IIABMan was phosphorylated for 5 min (pulse). At time 0, IICMan/IIDMan, 0.5 mm Glc, and a 50-fold molar excess of unlabeled PEP (chase) was added. As control, Glc and PEP but no IICMan/IIDMan were added to wild-type IIABMan (solid circles, dotted). The reactions were stopped at the indicated time points as described in A.D, same as C with the wild-type and the slow R168Q mutant but with different concentrations of IICMan/IIDMan (1 μg (dotted); 2 μg (dash-dotted); 4 μg (dashed); 8 μg (solid)) during the chase period. Wild-type (○); R168Q (□); R172Q (▵); R190K (▿); R204K (⬡); R304Q (♦). The maximum ratio of 32Pi to IIABMan is 2 because there are two phosphorylation sites per IIABMansubunit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The IIABMan subunit together with the transmembrane IICMan-IIDMan complex mediate transport and phosphorylation of mannose and related hexoses. Arginines play a specific role in many phosphoproteins where they stabilize the phosphoryl group in the ground and/or in the transition state (6Johnson L.N. Barford D. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 199-232Crossref PubMed Scopus (219) Google Scholar). The IIB proteins of E. coli, B. subtilis, andK. pneumoniae share five invariant arginines. To elucidate the functional role of these conserved residues for phosphorylation and transport, they were replaced one at a time with either Lys or Gln. Substrate phosphorylation is completely blocked in the R172Q mutant, strongly inhibited in the R168Q mutant, and three times slower than wild-type for the other mutants. Arg-172 together with His-175 is at the center of the active site loop (Fig.4). The conformation of Arg-172 in the crystal structure (of the nonphosphorylated IIBLev) precludes a direct interaction between the guanidino group and a phosphoryl group, which would be bound to Nδ of His-175. However, already a minor conformational change would suffice to bring the guanidino group within van der Waals distance of P-His-175. The high crystallographic B-factors for the loop and the complete absence of electron density for the Arg-172 side chain indicate flexibility allowing for such a move (20Schauder S. Nunn R.S. Lanz R. Erni B. Schirmer T. J. Mol. Biol. 1998; 276: 591-602Crossref PubMed Scopus (38) Google Scholar). Arg-168 and Arg-304 are components of a hydrogen bonding network that links the active site loop to a second large loop comprising residues 305–311. Other components of this network are the invariant Asp-170 of the active site loop, the invariant Asn-264 of the adjacent β-strand 5, residue 302 (Glu/Asp) of β-strand 6, and the invariant Asp-309 in the loop between β-strands 6 and 7 (Fig. 4). The β6/β7 loop is accessible from the protein surface and according to the docking model not involved in contacts to the IIA domain (20Schauder S. Nunn R.S. Lanz R. Erni B. Schirmer T. J. Mol. Biol. 1998; 276: 591-602Crossref PubMed Scopus (38) Google Scholar). Arg-204 is located on the edge of the first half of the β-sheet and does not interact directly with the phosphorylation site. The R204K mutant supports nonvectorial glucose phosphorylation but not glucose translocation. Arg-204 of IIABMan therefore appears important for solute translocation by the IICMan-IIDMan complex. Mutation of the nearby His-219 (H219Q) reduces the affinity of IIABMan for the IICMan-IIDMan complex resulting in 20-fold reduced phosphotransferase activity (25Stolz B. Huber M. Markovic-Housley Z. Erni B. J. Biol. Chem. 1993; 268: 27094-27099Abstract Full Text PDF PubMed Google Scholar). Finally, these residues and the region of the β-sheet comprising them yielded only weak signals in the NMR experiments with the isolated IIBMan domain. The high degree of conformational flexibility of this region could be due to missing interactions with the IICMan-IIDMancomplex (33Gschwind R.M. Gemmecker G. Leutner M. Kessler H. Gutknecht R. Lanz R. Flükiger K. Erni B. FEBS Lett. 1997; 404: 45-50Crossref PubMed Scopus (7) Google Scholar). Taken together these observations suggest that the first half of the β-sheet of IIBMan might form the interface with the IICMan-IIDMan complex. While this work was in progress four new transporters belonging to the mannose family were discovered, one for glucose in Lactobacillus curvatus (34Veyrat A. Gosalbes M.J. Pérez-Martı́nez G. Microbiology. 1996; 142: 3469-3477Crossref PubMed Scopus (16) Google Scholar), one for glucose and mannose in Vibrio furnissii (35Bouma C.L. Roseman S. J. Biol. Chem. 1996; 271: 33457-33467Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and a system for N-acetylgalactosamine in E. coli comprising two IIB subunits (IIBAgaand IIBAga′) (36Reizer J. Ramseier T.M. Reizer A. Charbit A. Saier Jr., M.H. Microbiology. 1996; 142: 231-250Crossref PubMed Scopus (62) Google Scholar). All five arginines are conserved inL. curvatus. In the IIBs of V. furnissii and of the E. coli GalNAc system only the two arginines near the active site histidine (Arg-168 and Arg-172) are invariant, whereas Ile and Leu are found in the position of Arg-190 and Gln instead of Arg-204 and Arg-304. Two Arg mutants (R426K and R428K) that have the same phenotype as R172Q of IIABMan were found in IICBGlc of theE. coli glucose transporter, which belongs to the glucose family and is structurally unrelated to the mannose transporter (12Lanz R. Erni B. J. Biol. Chem. 1998; 273: 12239-12243Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). When the phosphorylation cascade (enzyme I-HPr-IIA-IIB) of the two phosphotransferase systems for glucose and mannose are compared, the following patterns of structural and functional properties can be recognized. The active sites of enzyme I and IIA are on concave surfaces, they do not contain arginines near the active site, and the histidines are phosphorylated at Nε of the imidazole ring. The even numbered components of HPr and IIB have active sites protruding from the surface of the proteins, they are phosphorylated at Nδ of the imidazole ring or at a cysteine, and they contain one or several invariant and essential arginines. This periodicity and the structural and functional complementarity between alternating active sites is particularly striking between HPr and the IIB domain, where the conformations of the β/α active site loops are very similar (20Schauder S. Nunn R.S. Lanz R. Erni B. Schirmer T. J. Mol. Biol. 1998; 276: 591-602Crossref PubMed Scopus (38) Google Scholar). The fact that mutants can be phosphorylated but not donate the phosphoryl group to the next component suggests that the structural requirements for accepting a phosphoryl group are less exacting than for donating a phosphoryl group. The transition state apparently is stabilized by the phosphoryl donor rather than the phosphoryl acceptor protein. This was demonstrated with short basic peptides containing either a histidine or a cysteine that are rapidly phosphorylated by enzyme I but cannot donate the phosphoryl group further to an acceptor protein (37Mukhija S. Erni B. Mol. Microbiol. 1997; 25: 1159-1166Crossref PubMed Scopus (28) Google Scholar). The inverse mechanism has been observed with CheY, the response regulator of the chemotactic signaling cascade. CheY, which is phosphorylated at an aspartyl residue can autocatalyze the phosphoryl transfer not only from the cognate phosphoryl donor, the sensor kinase CheA, but also from low molecular weight phosphates such as phosphoramidate or acetylphosphate (38Lukat G.S. McCleary W.R. Stock A.M. Stock J.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 718-722Crossref PubMed Scopus (409) Google Scholar). Attempts to phosphorylate IIABMan with phosphohistidine or phosphoramidate failed so far. It remains to be seen in the future whether the phosphotransfer mechanisms of the PTS and of the two component regulatory systems (39Parkinson J.S. Kofoid E.C. Annu. Rev. Genet. 1992; 26: 71-112Crossref PubMed Scopus (1241) Google Scholar) can be characterized as catalysis by phospho-donor (kinase-like) and catalysis by phospho-acceptor (phosphatase-like), respectively. We thank Stephan Schauder for helpful discussions, suggestions, and the preparation of Fig. 4." @default.
• W1997495869 created "2016-06-24" @default.
• W1997495869 creator A5012264843 @default.
• W1997495869 creator A5037744451 @default.
• W1997495869 creator A5049957563 @default.
• W1997495869 date "1998-05-01" @default.
• W1997495869 modified "2023-10-03" @default.
• W1997495869 title "Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coliPhosphotransferase System" @default.
• W1997495869 cites W1497575646 @default.
• W1997495869 cites W1500422783 @default.
• W1997495869 cites W1531787215 @default.
• W1997495869 cites W1546793327 @default.
• W1997495869 cites W1558968590 @default.
• W1997495869 cites W1603465668 @default.
• W1997495869 cites W1604223977 @default.
• W1997495869 cites W1858328737 @default.
• W1997495869 cites W1957635815 @default.
• W1997495869 cites W1975330912 @default.
• W1997495869 cites W1984813978 @default.
• W1997495869 cites W1993105242 @default.
• W1997495869 cites W2001985302 @default.
• W1997495869 cites W2017256978 @default.
• W1997495869 cites W2028231353 @default.
• W1997495869 cites W2037955785 @default.
• W1997495869 cites W2039702459 @default.
• W1997495869 cites W2048019492 @default.
• W1997495869 cites W2048205348 @default.
• W1997495869 cites W2054158996 @default.
• W1997495869 cites W2060194246 @default.
• W1997495869 cites W2060913542 @default.
• W1997495869 cites W2070581282 @default.
• W1997495869 cites W2071001783 @default.
• W1997495869 cites W2072860601 @default.
• W1997495869 cites W2075066450 @default.
• W1997495869 cites W2075183488 @default.
• W1997495869 cites W2079357395 @default.
• W1997495869 cites W2079852902 @default.
• W1997495869 cites W2088587825 @default.
• W1997495869 cites W2108141139 @default.
• W1997495869 cites W2130394726 @default.
• W1997495869 cites W2146220380 @default.
• W1997495869 cites W2151007476 @default.
• W1997495869 cites W2157821159 @default.
• W1997495869 cites W2175146729 @default.
• W1997495869 cites W2178587031 @default.
• W1997495869 doi "https://doi.org/10.1074/jbc.273.20.12234" @default.
• W1997495869 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9575172" @default.
• W1997495869 hasPublicationYear "1998" @default.
• W1997495869 type Work @default.
• W1997495869 sameAs 1997495869 @default.
• W1997495869 citedByCount "12" @default.
• W1997495869 countsByYear W19974958692012 @default.
• W1997495869 countsByYear W19974958692013 @default.
• W1997495869 countsByYear W19974958692022 @default.
• W1997495869 crossrefType "journal-article" @default.
• W1997495869 hasAuthorship W1997495869A5012264843 @default.
• W1997495869 hasAuthorship W1997495869A5037744451 @default.
• W1997495869 hasAuthorship W1997495869A5049957563 @default.
• W1997495869 hasBestOaLocation W19974958691 @default.
• W1997495869 hasConcept C104292427 @default.
• W1997495869 hasConcept C104317684 @default.
• W1997495869 hasConcept C185592680 @default.
• W1997495869 hasConcept C54355233 @default.
• W1997495869 hasConcept C547475151 @default.
• W1997495869 hasConcept C70721500 @default.
• W1997495869 hasConcept C86803240 @default.
• W1997495869 hasConceptScore W1997495869C104292427 @default.
• W1997495869 hasConceptScore W1997495869C104317684 @default.
• W1997495869 hasConceptScore W1997495869C185592680 @default.
• W1997495869 hasConceptScore W1997495869C54355233 @default.
• W1997495869 hasConceptScore W1997495869C547475151 @default.
• W1997495869 hasConceptScore W1997495869C70721500 @default.
• W1997495869 hasConceptScore W1997495869C86803240 @default.
• W1997495869 hasIssue "20" @default.
• W1997495869 hasLocation W19974958691 @default.
• W1997495869 hasOpenAccess W1997495869 @default.
• W1997495869 hasPrimaryLocation W19974958691 @default.
• W1997495869 hasRelatedWork W1989493465 @default.
• W1997495869 hasRelatedWork W1991523530 @default.
• W1997495869 hasRelatedWork W2002128513 @default.
• W1997495869 hasRelatedWork W2020824267 @default.
• W1997495869 hasRelatedWork W2031436818 @default.
• W1997495869 hasRelatedWork W2057739827 @default.
• W1997495869 hasRelatedWork W2075354549 @default.
• W1997495869 hasRelatedWork W2080910126 @default.
• W1997495869 hasRelatedWork W2119103177 @default.
• W1997495869 hasRelatedWork W2092874662 @default.
• W1997495869 hasVolume "273" @default.
• W1997495869 isParatext "false" @default.
• W1997495869 isRetracted "false" @default.
• W1997495869 magId "1997495869" @default.
• W1997495869 workType "article" @default.