Rate and equilibrium constants for phosphoryltransfer between active site histidines of Escherichia coli HPr and the signal transducing protein IIIGlc

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) plays a central role in catabolizing many sugars; regulation is effected by phosphorylation of PTS proteins. In Escherichia coli, the phosphoryltransfer sequence for glucose uptake is: PEP --> Enzyme I(His191) --> HPr(His15) --> IIIGlc(His90) --> IIGlc(Cys421) --> glucose. A rapid quench method has now been developed for determining the rate and equilibrium constants of these reactions. The method was validated by control experiments, and gave the following results for phosphoryltransfer between the following protein pairs. For phospho-HPr/IIIGlc (and HPr/phospho-IIIGlc), k1 = 6.1 x 10(7) M-1 s-1, k-1 = 4.7 x 10(7); for the mutant H75QIIIGlc in place of IIIGlc, k1 = 2.8 x 10(5) M-1 s-1, k-1 = 2.3 x 10(5). The derived Keq values agreed with the Keq obtained without use of the rapid quench apparatus. Keq for both reactions is 1-1.5. The rate of phosphoryltransfer between HPr and wild type IIIGlc is close to a diffusion-controlled process, while the reactions involving the mutant H75QIIIGlc are 200-fold slower. These rate differences are explained by an hypothesis for the mechanism of phosphoryltransfer between HPr and IIIGlc based on the structures of mutant and wild type proteins (see Pelton et al. (Pelton, J. G., Torchia, D. A., Remington, S. J., Murphy, K. P., Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33446-33456)).     

The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic chitodextrinase

Chitin catabolism in Vibrio furnissii comprises several signal transducing systems and many proteins. Two of these enzymes are periplasmic and convert chitin oligosaccharides to GlcNAc and (GlcNAc)2. One of these unique enzymes, a chitodextrinase, designated EndoI, is described here. The protein, isolated from a recombinant Escherichia coli clone, exhibited (via SDS-polyacrylamide gel electrophoresis) two enzymatically active, close running bands ( approximately mass of 120 kDa) with identical N-terminal sequences. The chitodextrinase rapidly cleaved chitin oligosaccharides, (GlcNAc)4 to (GlcNAc)2, and (GlcNAc)5,6 to (GlcNAc)2 and (GlcNAc)3. EndoI was substrate inhibited in the millimolar range and was inactive with chitin, glucosamine oligosaccharides, glycoproteins, and glycopeptides containing (GlcNAc)2. The sequence of the cloned gene indicates that it encodes a 112,690-kDa protein (1046 amino acids). Both proteins lacked the predicted N-terminal 31 amino acids, corresponding to a consensus prokaryotic signal peptide. Thus, E. coli recognizes and processes this V. furnissii signal sequence. Although inactive with chitin, the predicted amino acid sequence of EndoI displayed similarities to many chitinases, with 8 amino acids completely conserved in 10 or more of the homologous proteins. There was, however, no "consensus" chitin-binding domain in EndoI.     

Topology of allosteric regulation of lactose permease

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.     

Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli

A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli. This putative P. furiosus PEP synthetase is slightly larger than the E. coli enzyme, the region between residues 58 and 89 being absent from the latter.     

Phosphorylation and functional properties of the IIA domain of the lactose transport protein of Streptococcus thermophilus

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIAGlc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIALacS would be able to perform one or more functions associated with IIAGlc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIALacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIALacS was unable to restore glucose uptake in a IIAGlc-deficient strain, which is consistent with a very low rate of phosphorylation of IIALacS by phosphorylated HPr (HPr approximately P) from E. coli. With HPr approximately P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIALacS (k1 = 4.3 x 10(2) M-1 s-1) and dephosphorylation of IIALacS approximately P by HPr (k-1 = 1.1 x 10(3) M-1 s-1) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIAGlc and HPr from E. coli. This finding suggests that IIALacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIAGlc, we constructed a double mutant [IIALacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIALacS(I548E/G556D) by HPr approximately P increased (k1 = 4.0 x 10(3) M-1 s-1) and became nearly independent of the source of HPr approximately P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIALacS(I548E/G556D) was insufficient to complement IIAGlc in PTS-mediated glucose transport in E. coli. Both IIALacS and IIALacS(I548E/G556D) could replace IIAGlc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.     

Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria

Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar. In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium. One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15. In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation. A series of mutations were made at Ser46 in E. coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly. The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria. In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%). The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values. The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation. There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure. There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr. In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11. The consequence of this change is that there is no torsion angle strain at residue 16. This result suggests that there is no active site torsion angle strain in wild-type E. coli HPr. The lack of substantial change at the active center of E. coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.     

The HPr(Ser) kinase of Streptococcus salivarius: purification, properties, and cloning of the hprK gene

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 microM and 1 mM, respectively.     

Binding of monoclonal antibody 4B1 to homologs of the lactose permease of Escherichia coli

The conformationally sensitive epitope for monoclonal antibody (mAb) 4B1, which uncouples lactose from H+ translocation in the lactose permease of Escherichia coli, is localized in the periplasmic loop between helices VII and VIII (loop VII/VIII) on one face of a short helical segment (Sun J, et al., 1996, Biochemistry 35;990-998). Comparison of sequences in the region corresponding to loop VII/VIII in members of Cluster 5 of the Major Facilitator Superfamily (MFS), which includes five homologous oligosaccharide/H+ symporters, reveals interesting variations. 4B1 binds to the Citrobacter freundii lactose permease or E. coli raffinose permease with resultant inhibition of transport activity. Because E. coli raffinose permease contains a Pro residue at position 254 rather than Gly, it is unlikely that the mAb recognizes the peptide backbone at this position. Consistently, E. coli lactose permease with Pro in place of Gly254 also binds 4B1. In contrast, 4B1 binding is not observed with either Klebsiella pneumoniae lactose permease or E. coli sucrose permease. When the epitope is transferred from E. coli lactose permease (residues 245-259) to the sucrose permease, the modified protein binds 4B1, but the mAb has no significant effect on sucrose transport. The studies provide further evidence that the 4B1 epitope is restricted to loop VII/VIII, and that 4B1 binding induces a highly specific conformational change that uncouples substrate and H+ translocation.     

Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE)

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]-[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.     

Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.     

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.     

Isolation and characterization of the recA gene of Xanthomonas campestris pv. campestris

A 1.8-kb NsiI-StuI fragment containing the recA gene of Xanthomonas campestris pv. campestris was cloned by a PCR-based approach and complementation of Escherichia coli HB 101. Sequence analysis of this fragment revealed an ORF (orf343) of 1,032 bp able to encode a protein of 343 amino acids with a calculated MW of 37,021 Da, a size similar to the values detected by in vitro system and Western blotting. It showed 69.6% identity to the E. coli RecA in amino acid sequence. Amino acid residues of the E coli RecA associated with functional activities are conserved in this Xc17 RecA. The recA mutant, L1, constructed by gene replacement, was sensitive to ultraviolet irradiation and methyl methanesulfonate, and deficient in homologous recombination.     

Profiles of published social work practitioners: who wrote and why

Pasteurella multocida was examined for glucose and mannose transport. P. multocida was shown to possess a phosphoenolpyruvate (PEP):mannose phosphotransferase system (PTS) that transports glucose as well as mannose and was functionally similar to the Escherichia coli mannose PTS. Phosphorylated proteins with molecular masses similar to those of E. coli mannose PTS proteins were visualized when incubated with 32P-PEP. The presence of an enzyme IIAGlc which could play an important role in regulation, as described in other Gram-negative bacteria, was detected. The enzymes of the pentose-phosphate pathway were present in P. multocida growth on glucose. The activity of 6-phosphofructokinase (the key enzyme of the Embden-Meyerhof pathway (EMP)), was very low in cell extracts, suggesting that EMP is not the major pathway for glucose catabolism.     

Fate of the upper urinary tract in multiple sclerosis

The Staphylococcus aureus 8325-4 hyaluronate lyase gene (hysA) was identified after detecting hyaluronate lyase activity expressed by phages from a genomic library. The hysA open reading frame, capable of encoding a protein of 91 980 Da, was identified by Tn5 mutagenesis and nucleotide sequencing. HysA shares 35 and 36% amino acid sequence identity with group B streptococcal hyaluronate lyase and pneumococcal hyaluronidase, respectively. A 94-kDa protein was expressed in Escherichia coli minicells, a result consistent with the coding capacity of hysA. Identification of the S. aureus 8325-4 hyaluronate lyase gene will allow the regulation of this putative virulence determinant to be studied.     

UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from Dictyostelium discoideum recognizes serine-containing peptides and eukaryotic cysteine proteinases

Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins. Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs. We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues. Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them. Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency. Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase. CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM. SDS denaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all. Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc-1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, Km = 5.6 microM. Thus, Ser:GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs. This may be physiologically important for establishing or maintaining non-overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium proteins that reside in functionally distinct endo-lysosomal vesicles.     

Relative contributions of subcutaneous and intermuscular fat to yields and predictability of retail product, fat trim, and bone in beef carcasses

Previous studies have suggested that the phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius consists of a nonphosphorylated enzyme II domain that functions in tandem with a separate enzymatic complex called III(Man). The III(Man) complex is believed to be composed of two protein dimers with molecular masses of approximately 72 kDa. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that one dimer is composed of two 38.9-kDa subunits called IIIH(Man), and the other of two 35.2-kDa subunits called IIIL(Man). This study was undertaken to determine (1) the number and nature of the phosphorylated residue(s) on IIIH(Man) and IIIL(Man) and the phosphorylation sequence allowing the transfer of the phosphoryl group from HPr(His approximately P) to the mannose:PTS substrates; (2) whether IIIH(Man) and IIIL(Man) originate from two different genes or result from a posttranslational modification; and (3) whether these two proteins are involved in the phosphorylation of 2-deoxyglucose, a substrate of the phosphoenolpyruvate:mannose phosphotransferase system. We showed that both IIIH(Man) and IIIL(Man) were phosphorylated on two histidine residues. One phosphate bond was heat-labile (phosphorylation at the N1 position of the imidazole ring), while the second was heat-resistant (phosphorylation at the N3 position of the imidazole ring). The sequence of the first phosphorylation site was deduced by comparing the N-terminal amino acid sequence of both forms of III(Man) with IIA domains of the EII-mannose family. The sequences of both forms were identical over the 15 first amino acids, that is, MIGIIIASHGKFAEG. The sequence of the second phosphorylation site was determined for IIIL(Man) as IHGQVATNxTP. Hence, IIIH(Man) and IIIL(Man) are PTS proteins of the IIAB type and should be renamed IIABH(Man) and IIABL(Man). IIABH(Man) and IIABL(Man) had different peptide profiles after digestion with proteases, indicating that these two proteins are encoded by two different genes. In vitro PEP-dependent phosphorylation assays conducted with a spontaneous mutant devoid of both forms of IIAB(Man) suggested that the phosphoenolpyruvate:mannose phosphotransferase system of S. salivarius is composed of an uncharacterized nonphosphorylated membrane component that works in tandem with IIABL(Man). The physiological functions of IIABH(Man) remain unknown.     

Molecular definition of breakpoints associated with human Xq isochromosomes: implications for mechanisms of formation

Inadequate knowledge of pathogenesis and pathophysiology has contributed to the high mortality and morbidity associated with neonatal Escherichia coli meningitis. We have shown previously that outer membrane protein A (OmpA) contributes to E. coli K1 membrane invasion of brain microvascular endothelial cells. In this study we report that this OmpA+ K1 E. coli invasion of brain microvascular endothelial cells was inhibited by wheat germ agglutinin and chitooligomers prepared from the polymer of 1,4-linked GlcNAc, chitin. The specificity of the interaction between OmpA and GlcNAc beta 1-4GlcNAc epitopes was verified by the demonstration that chitotriose-bound OmpA and wheat germ agglutinin-bound brain microvascular endothelial cell membrane proteins inhibit E. coli K1 invasion. Of interest, OmpA+ E. coli invasion into systemic endothelial cells did not occur, but invasion similar to that of brain microvascular endothelial cells was observed when systemic cells were treated with alpha-fucosidase, suggesting that the GlcNAc beta 1-4GlcNAc moieties might be substituted with L-fucose on these cells. More importantly, the chitooligomers prevented entry of E. coli K1 into the cerebrospinal fluid of newborn rats with experimental hematogenous E. coli meningitis, suggesting that the GlcNAc beta 1-4GlcNAc epitope of brain microvascular endothelial cells indeed mediates the traversal of E. coli K1 across the blood-brain barrier. A novel strategy with the use of soluble receptor analog(s) may be feasible in the prevention of devastating neonatal E. coli meningitis.     

Molecular heterogeneity of phospholipase D (PLD). Cloning of PLDgamma and regulation of plant PLDgamma, -beta, and -alpha by polyphosphoinositides and calcium

Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, vesicle trafficking, and membrane metabolism. This report describes the cloning and expression of a new Arabidopsis PLD cDNA, designated PLDgamma, and the regulation of PLDgamma, -beta, and -alpha by phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+. The PLDgamma cDNA is 3.3 kilobases in length and codes for an 855-amino acid protein of 95,462 Da with a pI of 6.9. PLDgamma shares a 66% amino acid sequence identity with PLDbeta, but only a 41% identity with PLDalpha. A potential N-terminal myristoylation site is found in PLDgamma, but not in PLDalpha and -beta. Catalytically active PLDgamma was expressed in Escherichia coli, and its activity requires polyphosphoinositides. Both PLDgamma and -beta are most active at microM Ca2+ concentrations, whereas the optimal PLDalpha activity requires mM Ca2+ concentrations. Binding studies showed that the PLDs bound PIP2 in the order of PLDbeta > PLDgamma > PLDalpha. This binding ability correlates with the degree of conservation of a basic PIP2-binding motif located near the putative catalytic site. The binding of [3H]PIP2 was saturable and could be competitively decreased by addition of unlabeled PIP2. Neomycin inhibited the activities of PLDgamma and -beta, but not PLDalpha. These results demonstrate that PLD is encoded by a heterogeneous gene family and that direct polyphosphoinositide binding is required for the activities of PLDgamma and -beta, but not PLDalpha. The different structural and biochemical properties suggest that PLDalpha, -beta, and -gamma are regulated differently and may mediate unique cellular functions.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EIIMan) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EIIMan showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EIIGlc). Similar homology was shown between the C-terminal sequence of EIIMan and the E. coli glucose-specific enzyme III (EIIIGlc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EIIMan contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIIIGlc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.