Sugar permeases of the bacterial phosphoenolpyruvate-dependent phosphotransferase system: sequence comparisons

The amino acyl sequences of eight permeases (enzymes II and enzyme II-III pairs) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) have been analyzed. All systems show similar sizes, and six of these systems exhibit the same molecular weight +/- 2%. Several exhibit sequence homology. Characteristic NH2-terminal and COOH-terminal sequences were found. The NH2-terminal leader sequences are believed to function in targeting of the permeases to the membrane, whereas the characteristic COOH-terminal sequences are postulated to mediate interaction with the energy-coupling protein phospho HPr. One of the systems, the one specific for mannose, exhibits distinctive characteristics. A pair of probable phosphorylation sites was detected in each of the five most similar systems, those specific for beta-glucosides, sucrose, glucose, N-acetylglucosamine, and mannitol. One of the two equivalent phosphorylation sites (proposed phosphorylation site 1) was located approximately 80 residues from the COOH terminus of each system. The other site (proposed phosphorylation site 2) was located approximately 440 residues from the COOH termini of the glucose and N-acetylglucosamine systems, approximately 320 residues from the COOH termini of the beta-glucoside and sucrose systems, and 381 residues from the COOH terminus of the mannitol system. Intragenic rearrangement during evolutionary history may account for the different positions of phosphorylation sites 2 in the different PTS permeases. More extensive intragenic rearrangements may have given rise to entirely different positions of phosphorylation in the glucitol, mannose, and lactose systems. A single, internal amphipathic alpha-helix with characteristic features was found in each of seven of the eight enzymes II. The lactose-specific enzyme III of Staphylococcus aureus was unique in possessing a COOH-terminal amphipathic alpha-helix rich in basic amino acyl residues. Possible functions for these amphipathic segments are discussed.     

Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants.

The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autophosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) catalysis of phosphoryl transfer to the IIA domains in these permeases.     

Carbohydrate Uptake and Utilization byClostridium beijerinckiiNCIMB 8052

The clostridia are a diverse group of obligately anaerobic bacteria with potential for the fermentative production of fuels, solvents and other chemicals. Several species exhibit a broad substrate range, but there have been few studies of the mechanisms involved in regulation of uptake and metabolism of fermentable carbohydrates.Clostridium beijerinckii(formerlyClostridium acetobutylicum) NCIMB 8052 exhibited transport activity for hexoses and hexitols. Glucose-grown cells transported glucose and fructose, but not galactose, glucitol (sorbitol) or mannitol, transport of which was induced by growth on the respective substrates. Phosphorylation of glucose, fructose, glucitol and mannitol by cell extracts was supported by phosphoenolpyruvate, indicating the involvement of a phosphotransferase system in uptake of these substrates. Fructose phosphorylation was also demonstrated by isolated membranes in the presence of fructose 1-phosphate, thus identifying this derivative as the product of the fructose phosphotransferase system. The presence of phosphotransferase activities in extracts prepared from cells grown on different carbon sources correlated with transport activities in whole cells, and the pattern of transport activities reflected the substrate preference of cells growing in the presence of glucose and another carbon source. Thus, glucose and fructose were co-metabolised, while utilization of glucitol was prevented by glucose, even in cells which were previously induced for glucitol metabolism. Of the substrates examined, only galactose appeared to be transported by a non-phosphotransferase mechanism, since a significant rate of phosphorylation of this sugar was supported by ATP rather than phosphoenolpyruvate.     

Reversal of the Mannitol-Sorbitol Diauxie in Escherichia coli

In Escherichia coli K-12 the proteins involved in the dissimilation of mannitol and sorbitol are specified by two separate gene clusters. The mannitol cluster appears to consist of a regulatory gene mtlC, a gene mtlA coding an enzyme II complex of the phosphoenolpyruvate phosphotransferase system, and a gene mtlD coding a mannitol-1-phosphate dehydrogenase. Three corresponding genes, sblC, sblA, and sblD, exist for the sorbitol pathway. In both pathways the hexitol captured from the medium and delivered into the cytoplasm as a phosphorylated compound is dehydrogenated to fructose-6-phosphate. The enzyme II complex for sorbitol is able to catalyze the phosphorylation also of mannitol if this substrate is present at high concentrations. Consequently mtlA(-) mutants lacking the enzyme II complex for mannitol can grow on mannitol either if the sorbitol phosphorylating system is preinduced by sorbitol or if mtlA is suppressed by a mutation of sblC to constitutivity. In wild-type cells, the induction of the enzymes in the mannitol pathway and dissimilation of the substrate are not prevented by glucose. The sorbitol system, however, is sensitive to glucose and to mannitol as well. In the suppressed strains (mtlA(-), sblC(c)) in which mannitol is utilized through the sorbitol enzyme, glucose becomes effective in restraining the consumption of mannitol, causing a definite diauxie. Moreover, in a mixture of mannitol and sorbitol, the latter is utilized preferentially. This reversal of normal diauxic pattern is consequent to the fact that the enzyme II complex for sorbitol has relatively poor affinity for mannitol.     

Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli.

The gut operon was subcloned into various plasmid vectors (M. Yamada and M. H. Saier, Jr., J. Bacteriol. 169:2990-2994, 1987). Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth. Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds. On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism. Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene. A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity. Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase. These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.     

Defective enzyme II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system leading to uncoupling of transport and phosphorylation in Salmonella typhimurium.

Transport and phosphorylation of glucose via enzymes II-A/II-B and II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system are tightly coupled in Salmonella typhimurium. Mutant strains (pts) that lack the phosphorylating proteins of this system, enzyme I and HPr, are unable to transport or to grow on glucose. From ptsHI deletion strains of S. typhimurium, mutants were isolated that regained growth on and transport of glucose. Several lines of evidence suggest that these Glc+ mutants have an altered enzyme II-BGlc as follows. (i) Insertion of a ptsG::Tn10 mutation (resulting in a defective II-BGlc) abolished growth on and transport of glucose in these Glc+ strains. Introduction of a ptsM mutation, on the other hand, which abolishes II-A/II-B activity, had no effect. (ii) Methyl alpha-glucoside transport and phosphorylation (specific for II-BGlc) was lowered or absent in ptsH+,I+ transductants of these Glc+ strains. Transport and phosphorylation of other phosphoenolpyurate:sugar phosphotransferase system sugars were normal. (iii) Membranes isolated from these Glc+ mutants were unable to catalyze transphosphorylation of methyl alpha-glucoside by glucose 6-phosphate, but transphosphorylation of mannose by glucose 6-phosphate was normal. (iv) The mutation was in the ptsG gene or closely linked to it. We conclude that the altered enzyme II-BGlc has acquired the capacity to transport glucose in the absence of phosphoenolpyruvate:sugar phosphotransferase system-mediated phosphorylation. However, the affinity for glucose decreased at least 1,000-fold as compared to the wild-type strain. At the same time the mutated enzyme II-BGlc lost the ability to catalyze the phosphorylation of its substrates via IIIGlc.     

Kinetic analyses of the sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose enzyme II complex of the bacterial phosphotransferase system.

The sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose Enzyme II complex of the phosphotransferase system has been analyzed kinetically. Initial rates of phosphoryl transfer from glucose-6-P to methyl alpha-glucopyranoside were determined with butanol/urea-extracted membranes from Salmonella typhimurium strains. The kinetic mechanism was shown to be Bi-Bi Sequential, indicating that the Enzyme II possesses nonoverlapping binding sites for sugar and sugar phosphate. Binding of the two substrates appears to occur in a positively cooperative fashion. A mutant with a defective glucose Enzyme II was isolated which transported methyl alpha-glucoside and glucose with reduced maximal velocities and higher Km values. In vitro kinetic studies of the transphosphorylation reaction catalyzed by the mutant enzyme showed a decrease in maximal velocity and increases in the Km values for both the sugar and sugar phosphate substrates. These results are consistent with the conclusion that a single Enzyme II complex catalyzes both transport and transphosphorylation of its sugar substrates.     

Nucleotide sequence of the fruA gene, encoding the fructose permease of the Rhodobacter capsulatus phosphotransferase system, and analyses of the deduced protein sequence.

The nucleotide sequence of the fruA gene, the terminal gene in the fructose operon of Rhodobacter capsulatus, is reported. This gene codes for the fructose permease (molecular weight, 58,575; 578 aminoacyl residues), the fructose enzyme II (IIFru) of the phosphoenolpyruvate-dependent phosphotransferase system. The deduced aminoacyl sequence of the encoded gene product was found to be 55% identical throughout most of its length with the fructose enzyme II of Escherichia coli, with some regions strongly conserved and others weakly conserved. Sequence comparisons revealed that the first 100 aminoacyl residues of both enzymes II were homologous to the second 100 residues, suggesting that an intragenic duplication of about 300 nucleotides had occurred during the evolution of IIFru prior to divergence of the E. coli and R. capsulatus genes. The protein contains only two cysteyl residues, and only one of these residues is conserved between the two proteins. This residue is therefore presumed to provide the active-site thiol group which may serve as the phosphorylation site. IIFru was found to exhibit regions of homology with sequenced enzymes II from other bacteria, including those specific for sucrose, beta-glucosides, mannitol, glucose, N-acetylglucosamine, and lactose. The degree of evolutionary divergence differed for different parts of the proteins, with certain transmembrane segments exhibiting high degrees of conservation. The hydrophobic domain of IIFru was also found to be similar to several uniport and antiport transporters of animals, including the human and mouse insulin-responsive glucose facilitators. These observations suggest that the mechanism of transmembrane transport may be similar for permeases catalyzing group translocation and facilitated diffusion.     

Structure and function of N-acetylglucosamine kinase. Identification of two active site cysteines.

N-Acetylglucosamine is a major component of complex carbohydrates. The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate degradation or nutritional sources starts with phosphorylation by N-acetylglucosamine kinase. In this study we describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction. Murine N-acetylglucosamine kinase contains six cysteine residues, all of which were mutated to serine residues. The strongest reduction of enzyme activity was found for the mutant C131S, followed by C143S. Determination of the kinetic properties of the cysteine mutants showed that the decreased enzyme activities were due to a strongly decreased affinity to either N-acetylglucosamine for C131S, or ATP for C143S. A secondary structure prediction of N-acetylglucosamine kinase showed a high homology to glucokinase. A model of the three-dimensional structure of N-acetylglucosamine kinase based on the known structure of glucokinase was therefore generated. This model confirmed that both cysteines are located in the active site of N-acetylglucosamine kinase with a potential role in the binding of the transferred gamma-phosphate group of ATP within the catalytic mechanism.     

Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon

The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene.     

Studies of the synthesis,structure and function of the phosphorylated oligosaccharides of lysosomal enzymes

Newly synthesized lysosomal enzymes were found to contain N-acetylglucosamine residues in phosphodiester linkage to the 6 position of the mannose residues on high-mannose type oligosaccharides. The formation of these structures was shown to be catalyzed by a specific N-acetylglucosaminylphosphotransferase enzyme, that utilises UDP-N-acetylglucosamine as a donor. The phosphorylation reaction can take place on any of four or five positions on the high-mannose oligosaccharide. Subsequently an α-N-acetylglucosaminylphosphodiesterase removes the outer blocking N-acetylglucosamine residues to generate the mature phosphomannsoyl recognition signal. This signal is responsible for the targetting of newly synthesized lysosomal enzymes to lysosomes. The human syndromes of I-cell disease (Mucolipidosis II) and pseudo-Hurler polydystrophy (Mucolipidosis III) were shown to be caused by deficiency of the first enzyme in the pathway, the UDP-N-acetylglucosamine: Glycoprotein N-acetylglucosaminylphosphotransferase.     

Analysis of the cooperativity of human beta-cell glucokinase through the stimulatory effect of glucose on fructose phosphorylation

Using overexpressed Escherichia coli sorbitol-6-phosphate dehydrogenase to monitor fructose 6-phosphate formation, we found that the stimulation of fructose phosphorylation by glucose was reduced in two human beta-cell glucokinase mutants with a low Hill coefficient or when the activity of wild type glucokinase was decreased by replacing ATP with poorer nucleotide substrates. Mutation of two other residues, neighboring glucose-binding residues in the catalytic site, also reduced the affinity for glucose as a stimulator of fructose phosphorylation. Among a series of glucose analogs, only 3, all substrates of glucokinase, stimulated fructose phosphorylation; other analogs were either inactive or inhibited glucokinase. Glucose increased the apparent affinity for inhibitors that are glucose analogs but not for the glucokinase regulatory protein or palmitoyl-CoA. These data indicate that the stimulatory effect of glucose on fructose phosphorylation reflects the positive cooperativity for glucose and is mediated by binding of glucose to the catalytic site. They support models involving the existence of two slowly interconverting conformations of glucokinase that differ through their affinity for glucose and for glucose analogs. We show by computer simulation that such a model can account for the kinetic properties of glucokinase, including the differential ability of mannoheptulose and N-acetylglucosamine to suppress cooperativity (Agius, L., and Stubbs, M. (2000) Biochem. J. 346, 413-421).     

Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease

The integral membrane protein responsible for the transport and phosphorylation of D-mannitol in Escherichia coli, the mannitol-specific Enzyme II of the phosphotransferase system (Mr = 60,000), has been purified to apparent homogeneity using a modification of a previously published procedure (Jacobson, G. R., Lee, C. A., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252). The purified enzyme was dependent on Lubrol PX and phospholipid for maximal activity. It catalyzed both the phosphoenolpyruvate- and the mannitol 1-phosphate-dependent phosphorylation of D-mannitol with high specificity for the accepting sugar and the phosphoryl donor. Both mannitol and mannitol 1-phosphate gave strong substrate inhibition at neutral pH in the transphosphorylation reaction catalyzed by the purified mannitol Enzyme II, while no substrate inhibition by mannitol was observed for the phosphoenolpyruvate-dependent reaction. The purified enzyme did not catalyze hydrolysis of mannitol 1-phosphate, a product of both reactions. Antibody directed against the mannitol Enzyme II inhibited the phosphoenolpyruvate-dependent activity to a greater extent than the transphosphorylation activity. Limited proteolysis with trypsin rapidly inactivated both purified and membrane-bound mannitol Enzyme II, and the purified protein was concomitantly cleaved into fragments with apparent molecular weights of about 29,000. These results show that although the mannitol Enzyme II is an integral membrane protein, a considerable portion of its polypeptide chain must also extend into a hydrophilic environment, presumably the cytoplasm.     

Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes. The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose and then purified by several ion exchange steps in the presence of the nonionic detergent, Lubrol PX. The purified protein appears homogeneous by several criteria and probably consists of a single kind of polypeptide chain with a molecular weight of 60,000 (+/- 5%). In addition to catalyzing phosphoenolpyruvate-dependent phosphorylation of mannitol in the presence of the soluble enzymes of the phosphotransferase system, the purified Enzyme II also catalyzes mannitol 1-phosphate:mannitol transphosphorylation in the absence of these components. A number of other physical and catalytic properties of the enzyme are described. The availability of a stable, homogeneous Enzyme II should be invaluable for studying the mechanism of sugar translocation and phosphorylation catalyzed by the bacterial phosphotransferase system.     

Hexitols as major intermediates of glucose assimilation by mycelium of Puccinia graminis

Mycelium of Puccinia graminis was grown for 4 d on 200 mM D-[U-¹⁴C]glucose followed by a cold chase for 30 h. Analysis of cellular metabolites during the chase indicated significant turnover only in carbohydrates soluble in 80% (w/v) ethanol. A kinetic analysis of the depletion of [¹⁴C] in pools of free sugars and sugar alcohols indicated that the trehalose pools and a small proportion (12–16%) of the mannitol and glucitol pools did not turn over, whilst pools of glucose, fructose, and the remainder of the hexitols became totally,depleted of label during the chase. Because the [¹⁴C] was totally lost from the pools of glucose and fructose prior to the hexitols, it was deduced that both of these hexoses were precursors of the hexitols. Estimation of the carbon fluxes through pools indicated that 52, 36 and 16% of the carbon from glucose was assimilated via glucitol, fructose and mannitol respectively, demonstrating that glucitol could not have originated from fructose as sole precursor. After offering D-[U-¹⁴C]glucitol, [¹⁴C] was assimilated into trehalose phosphate, glucans and amino acids, but not into free glucose or fructose. These data indicate that hexitols are quantitatively important intermediates during the assimilation of glucose by Puccinia graminis.     

Poly(C) synthesis by class I and class II CCA-adding enzymes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases], which catalyze synthesis of the conserved CCA sequence to the tRNA 3' end, are divided into two classes. Recent studies show that the class II Escherichia coli CCA-adding enzyme synthesizes poly(C) when incubated with CTP alone, but switches to synthesize CCA when incubated with both CTP and ATP. Because the poly(C) activity can shed important light on the mechanism of the untemplated synthesis of CCA, it is important to determine if this activity is also present in the class I CCA enzymes, which differ from the class II enzymes by significant sequence divergence. We show here that two members of the class I family, the archaeal Sulfolobus shibatae and Methanococcus jannaschii CCA-adding enzymes, are also capable of poly(C) synthesis. These two class I enzymes catalyze poly(C) synthesis and display a response of kinetic parameters to the presence of ATP similar to that of the class II E. coli enzyme. Thus, despite extensive sequence diversification, members of both classes employ common strategies of nucleotide addition, suggesting conservation of a mechanism in the development of specificity for CCA. For the E. coli enzyme, discrimination of poly(C) from CCA synthesis in the intact tRNA and in the acceptor-TPsiC domain is achieved by the same kinetic strategy, and a mutation that preferentially affects addition of A76 but not poly(C) has been identified. Additionally, we show that enzymes of both classes exhibit a processing activity that removes nucleotides in the 3' to 5' direction to as far as position 74.     

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis.

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of alpha-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of beta-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of beta-galactosidase activity in a strain carrying the ptsH1 mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsH1 mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsH1 mutant were also repressed in the ccpA mutant.     

Separation of four components of the phosphoenolpyruvate: glucose phosphotransferase system in Vibrio parahaemolyticus.

Four classes of Vibrio parahaemolyticus mutants defective in the phosphoenolpyruvate: glucose phosphotransferase system (PTS) are described. They were phenotypically different, and were defective in different PTS components. The components designated tentatively as II, I, III, and H were separated by gel filtration of a wild-type extract. Component II, which was specific for glucose and found in the particulate fraction, is probably membrane-bound, glucose-specific enzyme II. Both components I and H were soluble proteins, and the latter was relatively heat-stable. Component I was required for phosphorylation of glucose, trehalose, fructose, mannose, and mannitol. Component H was also required for phosphorylating all the above sugars except fructose. These and some additional findings strongly suggest that components I and H correspond to enzyme I and HPr, respectively. Component III, a soluble heat-stable protein, may be equivalent to the sugar-specific factor III found in other organisms, although it seems to participate in phosphorylating two sugars, glucose and trehalose. There were evidences that mutants defective in components I and III were deficient in cyclic adenosine 3',5'-monophosphate synthesis under certain conditions.     

The phosphoenolpyruvate-dependent carbohydrate: Phosphotransferase system enzymes II as chemoreceptors in chemotaxis of Escherichia coli K12

Summary In Escherichia coli K12, eight substrate-specific, membrane-bound enzymes II of the PEP-dependent carbohydrate: phosphotransferase system (PTS), specific for hexoses, hexosamines and hexitols, have been characterised in a series of isogenic and constitutive strains. In such mutants, lacking all but one enzyme II, the transport and vectorial phosphorylation activities as well as the chemotactical response in capillary tube assays have been compared. According to the data obtained, all enzymes II not only are directly involved in the transport and vectorial phosphorylation of their substrates, but they have also a primary role as the chemoreceptors for these substrates: (1) Metabolism of the attractant beyond the phosphorylation step is not a pre-requisite to eliciting positive chemotaxis. (2) Mutants, having only one enzyme II react in the capillary tube assay only to substrates of this enzyme II, but not to substrates of the missing enzymes II. This holds for enzymes II consisting of one membrane-bound protein as well as for systems containing a soluble factor III (FIII). (3) The substrate specificities or affinities, whether tested by transport and chemotaxis assays in vivo or by phosphorylation tests in vitro, are in correpondence. (4) The activities of enzymes II, regulated in a complex way at the level of enzyme synthesis and activity and tested as above, are also in agreement. (5) Mutants lacking the soluble proteins enzyme I or HPr of the PTS no longer respond chemotactically to any substrate taken up and phosphorylated by enzymes II. It is concluded that in PTS enzymes II some functions required for transport and chemotaxis are identical. It is suggested furthermore, that the alternation of intrinsic membrane-bound proteins between a phosphorylated and a dephosphorylated state, rather than binding of the substrate to the enzyme II, is the decisive stimulus in the chemotaxis toward carbohydrates taken up by these transport systems.     

Mucolipidoses II and III variants with normal N-acetylglucosamine 1-phosphotransferase activity toward alpha-methylmannoside are due to nonallelic mutations.

Normal N-acetylglucosamine 1-phosphotransferase activity toward mono- and oligosaccharide acceptor substrates was detected in cultured skin fibroblasts from mucolipidoses II and III patients who were designated as variants (one of four mucolipidosis II and three out of six mucolipidosis III patients examined). The activity toward natural lysosomal protein acceptors was absent or deficient in cell preparations from all patients with classical as well as variant forms of mucolipidoses II and III. Complementation analysis, using fused and cocultivated mutant fibroblast combinations, revealed that, while cell lines with variant mucolipidosis III constituted a complementation group distinct from that of classical forms of mucolipidoses II and III, the variant mucolipidosis II cell line belonged to the same complementation group as did the classical forms. In contrast to the mutant enzyme from variant mucolipidosis III patients that failed to recognize lysosomal proteins as the specific acceptor substrates, the activity toward alpha-methylmannoside in the variant mucolipidosis II patient could be inhibited by exogenous lysosomal enzyme preparations (bovine beta-glucuronidase and human hexosaminidase A). These findings suggest that N-acetylglucosamine 1-phosphotransferase is composed of at least two distinct polypeptides: (1) a recognition subunit that is defective in the mucolipidosis III variants and (2) a catalytic subunit that is deficient or altered in the classical forms of mucolipidoses II and III as well as in the mucolipidosis II variant.