Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coli Phosphotransferase System*

Regula Gutknecht, Regina Lanz, and Bernhard ErniDagger

From the Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (1). Protein kinases and protein phosphatases regulate the activity of enzymes and membrane bound receptors by phosphorylation and dephosphorylation (2, 3). In the phosphoenolpyruvate-dependent phosphotransferase system of bacteria (PTS)1 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. 4 and 5).

Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions (6). 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 (7). 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 beta -sheet with three alpha -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 (8). 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 (9). 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 (10, 11). 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 the E. coli IICBGlc subunit), and both are essential for phospho-donor activity toward glucose but not for phospho-acceptor activity (12). Five arginine residues, Arg-168, Arg-172, Arg-190, Arg-204, and Arg-304 (numbers refer to E. coli IIABMan subunit), are invariant in the IIB domains of the PTS transporters of E. coli, Klebsiella pneumoniae (13), and Bacillus subtilis (14) 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 (15). They contain the substrate binding site, and they facilitate the penetration of bacteriophage lambda  DNA by an as yet unknown mechanism (16, 17). Two IIABMan subunits form a stable homodimer, which reversibly binds to the IICMan-IIDMan complex (KD 5-10 nM). The IIABMan subunit consists of two protein domains (IIA and IIB), which are connected by a 20-residue-long hinge peptide rich in alanine and proline (18). 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. IIBMan then donates the phosphate to the sugar. Although IIBMan can 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 IIAMan domain has an alpha /beta open pleated sheet structure. The central beta -sheet consists of four parallel strands from one subunit and one antiparallel strand from the second subunit in the dimer (19). This beta  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 (20). It has an open pleated alpha /beta fold consisting of a strongly twisted seven-stranded beta -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 (20). Here we characterize the functions of the invariant arginines in the IIBMan domain of the mannose transporter.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Media, Strains, and Plasmids-- E. coli K12-UT580Delta LPM (Delta manXYZ::cat) and ZSC112LDelta LPM(Delta manXYZ::cat ptsG glk) carry a deletion of the chromosomal manXYZ allele. Gene replacement was done in E. coli DPE271 (recD) as described (21) with the following modifications. (i) The HindIII-KpnI fragment encoding residues 111-323 of IIABMan, the complete IICMan, and residues 1-241 of IIDMan was removed from plasmid pTSML1 (22) and replaced by KpnI-HindIII fragment from plasmid pMc5-8 (23) encoding cat (the KpnI site was introduced into the original pMc5-8 by gapped duplex site-directed mutagenesis). (ii) The recombinant plasmid pTSDelta LPM::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. ZSC112Delta LPM was transformed with plasmid pTSPM6 (encoding IICMan and IIDMan) (18) and a second plasmid encoding the IIABMan mutants, and the strains were used for in vivo experiments. WA2127Delta HIC (manXYZ, Delta ptsH ptsI crr) (24) 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.

Site-directed Mutagenesis-- IIABMan mutants R168Q,R172Q, R304Q were constructed using the gapped duplex procedure (23, 25). 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 (26). Mutant clones were identified by DNA sequencing. The HindIII-SnaBI segments carrying the mutations were cloned into the expression vector pJFL (encoding manX under the control of tacP, identical with pTACL293) (27).

Overproduction and Purification of Proteins-- E. coli WA2127Delta HIC was transformed with derivatives of pJFL encoding wild-type and mutant IIABMan. IIABMan was overexpressed and purified as described (25). Enzyme I and HPr were purified as described (24). The IICMan-IIDMan complex was purified by metal chelate affinity chromatography (28).2

Assay for Phosphotransferase Activity-- In vitro phosphorylation of [14C]Glc was assayed by ion-exchange chromatography as described (25). 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.

Transport Assay-- Uptake of [14C]dGlc by bacteria was assayed as described (29). The [14C]dGlc concentration was 120 µM, the specific activity was 6600 dpm/nmol. The cell density in the assay mixture was A600 = 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.

Assay for Protein Phosphorylation and Dephosphorylation-- The rate and the extent of protein phosphorylation and dephosphorylation was measured in a filter binding assay (21). The incubation mixture (50 mM NaPi, pH 7.4, 5 mM MgCl2, 2.5 mM NaF, 2.5 mM dithiothreitol) 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 (21). 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 (30).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

In Vivo Transport Activity-- 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 µM isopropyl-1-thio-beta -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. 1A). The arginine mutants R168Q, R172Q and R204K had less than 5% transport activity. The results from McConkey plates and in vivo transport 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 (25).


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Fig. 1.   Phosphotransferase activities of IIABMan. A, uptake of dGlc by intact cells expressing different IIABMan mutants was measured as described under "Experimental Procedures." B, Phosphorylation of Glc by purified IIABMan. IIABMan activity was titrated in the presence of saturating concentrations of enzyme I, HPr, and IICMan/IIDMan as described under "Experimental Procedures." Wild-type (open circle ); R168Q (square ); R172Q (triangle ); R190K (down-triangle); R204K (); R304Q (black-diamond ).

In Vitro Phosphotransferase Activity-- 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 (31, 32). 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-IIDMan complex, and the formation of [14C]Glc6P was measured (Fig. 1B). 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 low in vivo transport activity, retained 70% nonvectorial phosphorylation activity.

Protein Phosphorylation-- 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. 2A). The amount of 32P in the IIB band is lower than in the IIA band because histidine Ndelta phosphates are more sensitive to hydrolysis than histidine Nepsilon phosphates and because the IIB domain also is more sensitive to incipient trypsinolysis than IIA (18). The ratio 32P-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. 2B, + 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. 2B- lanes). This is possibly due to IICMan/IIDMan-mediated spontaneous hydrolysis, which is faster with active IIABMan than 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. 3A). 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. 3B). The dephosphorylation rates were measured in a pulse-chase experiment. IIABMan was labeled with 32P 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. 3D).


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Fig. 2.   Characterization of IIABMan by limited trypsinolysis and autoradiography. A, purified IIABMan was incubated with [32P]PEP in the presence of enzyme I and HPr for 10 min at 37 °C. One aliquot (+) was then treated with trypsin for 30 min to separate the IIA and the IIB domain. The reaction was stopped with trypsin inhibitor. The second aliquot was analyzed intact (-). Note that the R172Q mutant shows a cleavage pattern that differs reproducibly from the norm. The stronger bands comigrating with enzyme I are IIABMan dimers. The fainter bands between enzyme I and the IIABMan monomer (in the + lanes) are partially cleaved IIAB/IIA dimers. The double bands of IIB and IIA are the products of single and double cleavage after lysine 127 and 147 in the hinge peptide (18). B, purified IIABMan was incubated with [32P]PEP in the presence of enzyme I, HPr, and IICMan/IIDMan for 10 min. To one aliquot (+) a molar excess of Glc was added to dephosphorylate the PTS proteins. Note that the R172Q mutant is not dephosphorylated, and that wild-type, R190K, and R304Q are slowly dephosphorylated in the presence of IICMan/IIDMan even in the absence of Glc. W.t., wild type; EnzI, enzyme I.


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Fig. 3.   Time 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 A but 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 (open circle ); R168Q (square ); R172Q (triangle ); R190K (down-triangle); R204K (); R304Q (black-diamond ). The maximum ratio of 32Pi to IIABMan is 2 because there are two phosphorylation sites per IIABMan subunit.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (6). The IIB proteins of E. coli, B. subtilis, and K. 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 Ndelta 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 (20). 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 beta -strand 5, residue 302 (Glu/Asp) of beta -strand 6, and the invariant Asp-309 in the loop between beta -strands 6 and 7 (Fig. 4). The beta 6/beta 7 loop is accessible from the protein surface and according to the docking model not involved in contacts to the IIA domain (20).


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Fig. 4.   Structure of the IIBMan domain. Cartoon of the homology model derived from the x-ray structure of IIBLev (20). The active site His-175, and the invariant arginine residues are indicated. Arg-168 forms a salt bridge to Asp-170 and three H-bridges with the main chain carboxyl oxygens of Arg-304 and Gln-305 and with the side-chain oxygen of Asn-264. Arg-172 is located in the active site loop, which is disordered in the x-ray structure. Arg-190 is exposed on the protein surface on the opposite side of the active site and does not display contacts. Arg-204 forms a salt bridge to Glu-224. Arg-304 forms a salt bridge to Glu-302 and a H-bridge to the main chain carboxyl oxygen of Asp-309. The figure was produced using MOLSCRIPT (40).

Arg-204 is located on the edge of the first half of the beta -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 (25). Finally, these residues and the region of the beta -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-IIDMan complex (33). Taken together these observations suggest that the first half of the beta -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 (34), one for glucose and mannose in Vibrio furnissii (35), and a system for N-acetylgalactosamine in E. coli comprising two IIB subunits (IIBAga and IIBAga') (36). All five arginines are conserved in L. 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 the E. coli glucose transporter, which belongs to the glucose family and is structurally unrelated to the mannose transporter (12). 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 Nepsilon 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 Ndelta 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 beta /alpha active site loops are very similar (20). 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 (37). 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 (38). 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 (39) can be characterized as catalysis by phospho-donor (kinase-like) and catalysis by phospho-acceptor (phosphatase-like), respectively.

    ACKNOWLEDGEMENTS

We thank Stephan Schauder for helpful discussions, suggestions, and the preparation of Fig. 4.

    FOOTNOTES

* This work was supported by Grant 31-45838.95 from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 41-031-631-43-46; Fax: 41-0-31-631-48-87; E-mail:erni{at}ibc.unibe.ch.

1 The 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.

2 R. Gutknecht, R. Lanz, and B. Erni, unpublished results.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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