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PubMed1990

Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei. Evidence that a cysteine residue is essential for sugar phosphorylation.

The gene coding for the lactose-specific Enzyme II of the Lactobacillus casei phosphoenolpyruvate-dependent phosphotransferase system, lacE, has been isolated by molecular cloning and expressed in Escherichia coli. The DNA sequence of the lacE gene and the deduced amino acid sequence are presented. The putative translation product comprises a hydrophobic protein of 577 amino acids with a calculated molecular mass of 62,350 Da. The deduced polypeptide has a high degree of sequence similarity with the corresponding lactose-specific enzymes II of Staphylococcus aureus and Lactococcus lactis. The sequence surrounding cysteine 483 was strongly conserved in the three proteins. The identity of the lacE product as the Enzyme IIlacL.casei was demonstrated by in vitro lactose phosphorylation assays using the protein expressed in E. coli. Single replacement of each of the histidine and cysteine residues by site-directed mutagenesis pointed to cysteine 483 as an amino acid residue essential for the phosphoryl group transfer reaction.

Base Sequence↗

PubMed1990

Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase.

Antibodies against the alpha (Mr 67,000) and beta (Mr 60,000) subunits of wheat seedling Fru-2,6-P2-stimulated pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) were used to probe the subunit structures of several partially purified plant PFPs after tryptic digestion. Antisera to the alpha and beta subunits of wheat seedling PFP cross-reacted with the corresponding alpha and beta subunits of PFP preparations from wheat germ, potato tubers, and lettuce leaves. With the mung bean PFP, both antisera reacted with a protein band of Mr 60,000. A protein band corresponding to the Mr 67,000 alpha subunit was not detected in the mung bean PFP preparation. Tryptic digestion of wheat seedling and potato tuber PFPs resulted in the preferential cleavage of the alpha subunit. The trypsinized PFP retained most of its Fru-2,6-P2-stimulated activity but not its basal activity. The proteolyzed enzyme also exhibited a 2-fold increase in Ka for Fru-2,6-P2. Studies with the mung bean enzyme revealed that the anti-alpha immunoreactive component was more sensitive to trypsinization than the anti-beta immunoreactive component of the Mr 60,000 protein band. Thus, the Mr 60,000 protein band of the mung bean PFP appears to be heterogeneous and contains both alpha and beta-like proteins. The above observations indicate that the alpha and beta subunits of PFP are two distinct polypeptides and that alpha acts as a regulatory protein in regulating both the catalytic activity and the Fru-2,6-P2-binding affinity of the beta subunit.

Antibodies↗

PubMed1978

[Aminoglycoside-3'-phosphotransferase I from aminoglycoside-polyresistant strain E. coli 182].

An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.

Aminoglycosides↗

PubMed1990

Regulation of the maltose transport system of Escherichia coli by the glucose-specific enzyme III of the phosphoenolpyruvate-sugar phosphotransferase system. Characterization of inducer exclusion-resistant mutants and reconstitution of inducer exclusion in proteoliposomes.

Maltose transport in Escherichia coli is regulated at the protein level by the glucose-specific enzyme III (IIIglc) of the phosphoenolpyruvate-sugar phosphotransferase system, by a mechanism known as inducer exclusion. We have isolated and characterized four mutants in the maltose transport system, all of which are in malK, which are resistant to inducer exclusion. The mutations in three of these mutants fall within the COOH-terminal domain of MalK and suggest the first reported function for this domain. Two of these are in a region which shows sequence similarity to lacY and melB, both of which are also regulated by IIIglc, and thus may define a IIIglc-binding domain. We have also reconstituted inducer exclusion in proteoliposomes made from membranes overexpressing the maltose permease. Maltose transport is inhibited by 50-60% when IIIglc is included in the intravesicular space. The inhibition is due to a decrease in the Vmax of transport by a factor of 2. IIIglc does not affect the coupling of ATP hydrolysis to maltose transport, since the ratio of ATP hydrolyzed/maltose transported remained constant in the presence and absence of IIIglc. Finally, the Ki for IIIglc was 40 microM, roughly the same as the in vivo concentration of IIIglc.

Amino Acid Sequence↗

PubMed1989

Cloning and characterization of cDNA for mitochondrial GTP:AMP phosphotransferase of bovine liver.

Three different types of cDNA clones for mitochondrial GTP:AMP phosphotransferase (AK3) were isolated from a cDNA library of bovine liver poly(A)+ RNA. Nucleotide sequencing revealed that each of these clones consisted of a common 5'-untranslated region, a common AK3-coding sequence and a 3'-untranslated region with different sizes. By Northern blot analysis, three species of AK3 mRNA apparently corresponding to the isolated cDNA clones were detected, which would be a result of varying terminations and polyadenylations of the primary transcript. From comparison of the size of the product synthesized in vitro from the message directed by the isolated cDNA with that of the purified AK3 protein, AK3 appeared to have no cleavable NH2-terminal sequence as found in other mitochondrial proteins. The AK3 cDNA was expressed in Escherichia coli, which resulted in complementation of an adenylate kinase mutation of E. coli. The AK3 product was exported to the periplasmic space through the bacterial inner membrane. The possible involvement of the NH2-terminal sequence of the protein in targeting to the mitochondrial matrix was discussed.

Amino Acid Sequence↗

PubMed1989

Studies on adenylate kinase (ATP:AMP phosphotransferase) of Mycobacterium marinum (ATCC 927).

The enzyme adenylate kinase (ATP:AMP phosphotransferase) was purified as described previously [Biochim. Biophys. Acta 869: 350 (1986)] with an additional step involving affinity chromatography on Cibacron Blue. The molecular weight of the final enzyme preparation was estimated to be 22,500 on polyacrylamide-gel electrophoresis under denaturing conditions. The preliminary amino acid analysis indicated the presence of two histidine residues. Photooxidation in the presence of methylene blue caused complete inactivation of the enzyme, but the loss of activity could be prevented by the addition of ATP, AMP or adenosine-(5')-pentaphospho-(5')-adenosine, indicating that at least one histidine residue is involved at the active site. A circular dichroic study indicated that the enzyme consists of 24% alpha-helix, 30% beta-structure, and 46% random coil. The bacterial cells induced with antimycin A and light (particularly with the former) appeared to have somewhat increased adenylate kinase activity, although the Km and Vmax values were unchanged.

Adenine Nucleotides↗

PubMed1989

Crystallization of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli.

Single crystals of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli have been grown. This represents one of the first Fab-protein antigen complexes in which the same Fab fragment has previously been crystallized in the uncomplexed state and the structure solved (Prasad, L., Vandonselaar, M., Lee, J. S., and Delbaere, L. T. J. (1988) J. Biol. Chem. 263, 2571-2574). Single crystals up to 0.25 x 0.50 x 0.05 mm in size were grown by the technique of washing and reseeding. The space group is C2, with unit cell dimensions a = 130.0, b = 68.1, and c = 77.6 A; beta = 97.3 degrees; and Z = 4. There is one Fab-histidine-containing protein complex/asymmetric unit, and the solvent content is estimated to be 57%.

Antibodies, Monoclonal↗

PubMed1987

[Demonstration of a pyrophosphate-fructose 6-phosphate 1-phosphotransferase in the latex from Hevea brasiliensis].

A pyrophosphate: fructose-6-phosphate 1-phosphotransferase activity (EC 2.7.1.90) has been characterized in cytosol from Hevea brasiliensis latex. It is Mg+ dependent enzyme, and the cation has an optimal effect between 2.5 to 3 mM for a concentration of 1 mM of pyrophosphate and 10 mM of fructose-6-phosphate. It is activated by catalytic content of fructose-2,6-diphosphate. Its potential activity is higher than 40% of that of ATP dependent phosphofructokinase (EC 2.7.1.11). Its optimum pH is between 7.5-7.6; then, the enzyme affinity is 0.3 mM for pyrophosphate and 3.5 mM for fructose-6-phosphate. It is suggested that the transferase plays a role in the pyrophosphate metabolism and the increasing of the energetic efficiency of glycolysis and so takes a significant part in the biochemical mechanisms involved in the latex yield.

Cytosol↗

PubMed1987

Identification of the genes for the lactose-specific components of the phosphotransferase system in the lac operon of Staphylococcus aureus.

The nucleotide and deduced amino acid sequences of the lacE and lacF genes, which code for the lactose-specific Enzyme II and Enzyme III of the Staphylococcus aureus phosphotransferase system, are presented. The primary translation products consist of a hydrophobic protein of 572 amino acids (Mr = 62,688) and a polypeptide of 103 amino acids (Mr = 11,372), respectively. The assignment of lacF as the gene for Enzyme IIIlac was based upon the known amino acid sequence of the protein. The identity of lacE as encoding Enzyme IIlac was based upon immunoreactivity of the cloned gene product with antibodies raised against purified Enzyme IIlac from S. aureus and an assay of biological function of the protein expressed in Escherichia coli. The order of the known genes of the S. aureus lac operon is lacF-lacE-lacG, the latter encoding phospho-beta-galactosidase.

Amino Acid Sequence↗

PubMed1988

CTP: CMP phosphotransferase activity in L1210 cells.

Substrate converting enzymes interfering with the measurement of ribonucleotide reductase were assessed in cell-free extracts prepared from L1210 cells. Data show the presence of a myokinase-type enzyme activity (CTP:CMP phosphotransferase) which catalyzes the reaction: 2CDP in equilibrium CMP + CTP. This enzyme is not removed by passage of cell extracts over ATP-agarose columns. Monitoring of nucleoside diphosphate substrate level is, therefore, mandatory for obtaining accurate measurements of CDP reductase activity in crude cell extracts.

Animals↗

PubMed1988

[Aminoglycoside 3'-phosphotransferase gene: its cloning, characteristics and use as a molecular probe].

Aminoglycoside resistance genes were cloned from transconjugates of aminoglycoside resistant clinical strains of gramnegative bacteria. The resistance determinant cloned from the strains of E. coli transferred kanamycin, monomycin and neomycin resistance to laboratory strains. It was shown that the cloned resistance gene encoded the type I 3'-aminoglycoside phosphotransferase. The results of spot and blot hybridization of the gramnegative bacteria clinical strains with the Bam HI-Pst I fragment of the cloned resistance determinant were indicative of wide-spread distribution of the APH 3' (I) and closely related genes in clinical microbial populations.

Aminoglycosides↗

PubMed1987

[APH(3')-II phosphotransferase determination using protein transfer (western blotting)].

The APH(3')-II aminoglycoside-phosphotransferase has been studied by protein blotting. This technique allowed the detection of the enzyme in the crude extracts from clinical isolates of Enterobacteriaceae, and the molecular weight determination of the APH(3')-II. The obtained molecular weight of 25,000 is coincident with the previously reported value determined by a different method. These results demonstrate that this technique is useful for the study of aminoglycoside-modifying enzymes.

Blotting, Western↗

PubMed1987

Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes.

Specialized lambda-transducing phages that carry the Escherichia coli genes ptsH, ptsI, crr, cysM, and cysA have been isolated, and the genes were subcloned in plasmid pBR322. Subcloning and restriction mapping data gave the following clockwise order of genes located at about 52 min on the E. coli genetic map: lig, cysK, ptsH, ptsI, crr, cysM, cysA. The nucleotide sequences of ptsH, ptsI, and crr and the corresponding flanking regions have been determined. These genes encode three cytoplasmic proteins of the phosphoenol-pyruvate:glycose phosphotransferase system: HPr, Enzyme I, and IIIGlc, respectively. The deduced amino acid sequences are consistent with amino acid composition and Edman degradation analyses obtained with the purified proteins. The calculated subunit molecular weight values (9,109 for HPr, 63,489 for Enzyme I, and 18,099 for IIIGlc) also agree well with values obtained with the proteins. Results of gamma delta-transposon insertional studies provided definitive evidence that IIIGlc is the gene product of crr, and therefore that IIIGlc plays a critical role in regulating the metabolism and uptake of certain non-PTS sugars (see accompanying papers: Mitchell, W.J., Saffen, D.W., and Roseman, S. (1987) J. Biol. Chem. 16254-16260; Misko, T.P., Mitchell, W.J., Meadow, N.D., and Roseman, S. (1987) J. Biol. Chem. 16261-16266). The gamma delta transposon studies also suggest that crr is transcribed from an independent promoter located within the ptsI gene. Putative regulatory sequence features include a catabolite gene activator protein-cAMP-binding site and two regions of 2-fold rotational symmetry adjacent to the potential promoter upstream from the HPr structural gene, several ribosome-binding sites, and a rho-independent RNA polymerase termination site downstream from crr. In addition, the ptsI gene contains two highly conserved direct repeats. The significance of these sequence features is discussed with respect to possible multiple forms of pts regulation.

Amino Acid Sequence↗

PubMed1985

[Inhibitor of aminoglycoside phosphotransferases from Micromonospora sp.--a producer of sisomicin].

A substance inhibiting the activity of aminoglycoside-3-'-phosphotransferases of both the actinomycetous and the bacterial origin was detected in the filtrates of the culture fluid and mycelium of Micromonospora sp. producing sisomicin. The activity of the substance against the aminoglycoside inactivating enzymes of different types was studied. It was shown that the quantity of the inhibitor in the culture fluid of Micromonospora sp. correlated with intensity of sisomocin production. Under conditions not providing production of the antibiotic the inhibitory activity was lacking. The inhibitor was purified by ion exchange chromatography on Amberlite CG-50 and KM-cellulose (NH+4 form), gel filtration through Sefadex G-50 and preparative paper chromatography. Stability of the inhibitor at different pH values and different temperatures, its sensitivity to certain enzymes and behaviour in high voltage electrophoresis were studied. The results of ultrafiltration showed that the molecular weight of the inhibitor was relatively low: less than 1000 D.

Chromatography, Gel↗

PubMed1985

The mannose-permease of the bacterial phosphotransferase system. Gene cloning and purification of the enzyme IIMan/IIIMan complex of Escherichia coli.

The mannose-permease complex of the phosphoenolpyruvate-dependent phosphotranferase system exhibits two apparently unrelated activities. It mediates active transport concomitant with phosphorylation of mannose, 2-deoxyglucose, and a number of other hexoses, and it is required for penetration of bacteriophage lambda DNA across the cytoplasmic membrane of Escherichia coli. A cloned fragment of E. coli chromosome restores mannose-fermentation in, and confers lambda sensitivity to, an E. coli strain with a mutation in the gene for the phosphoenolpyruvate-dependent mannose uptake. Using complementation analysis of phosphotransferase activity and of lambda sensitivity, a 3.8-kilobase pair fragment was shown to carry two adjacent genes, ptsM and ptsL. Although each gene has a promoter of its own, transcription of ptsL has a positive polar effect on the transcription of ptsM. A complex of two proteins, IIMan and IIIMan, was purified to homogeneity from an overproducing strain. IIMan is encoded by gene ptsM, IIIMan by gene ptsL. IIMan, a 27-kDa protein, is the transmembrane component of the complex. IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm. IIIMan can be phosphorylated in a phosphoenolpyruvate-dependent reaction, while phosphorylation of IIMan could not be detected. IIMan and IIIMan are both required for phosphorylation of 2-deoxyglucose in vitro, while IIMan alone is sufficient to confer lambda sensitivity.

Antibodies, Monoclonal↗

PubMedDate not supplied

[Expression of aminoglycoside phosphotransferase gene is under the control of recA promoter].

A novel expression vector using the 300 bp promotor-operator fragment of the recA gene of Escherichia coli has been constructed. The strength of the recA promotor was examined by assaying aminoglycoside phosphotransferase (APT) activity expressed from APT gene placed downstream of the promotor. We have observed, that some plasmids, containing N-portion of recA gene caused a large increase in radiosensitivity of host bacteria cells.

Escherichia coli↗

PubMed1986

Glucose-permease of the bacterial phosphotransferase system. Gene cloning, overproduction, and amino acid sequence of enzyme IIGlc.

The glucose-permease (IIGlc) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It also functions as a receptor for bacterial chemotaxis. The structural gene of the permease, ptsG, has been cloned on a multicopy plasmid, and transformants constitutively overproducing the protein 10-15 times over wild-type level have been isolated. Overproduction is slightly inhibited if transformants are grown in a glucose-containing medium. The complete amino acid sequence of the glucose-permease is deduced from the nucleotide sequence. It consists of 477 residues and is moderately hydrophobic. A comparison of the glucose-permease with the mannitol-permease (Lee, C. A., and Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 10761-10767) does not reveal any obvious homology at the level of amino acid sequence.

Amino Acid Sequence↗

PubMed1988

DNA sequence of the gene scrA encoding the sucrose transport protein EnzymeII(Scr) of the phosphotransferase system from enteric bacteria: homology of the EnzymeII(Scr) and EnzymeII(Bgl) proteins.

The nucleotide sequence of the structural gene, scrA, which codes for sucrose-specific EnzymeII(Scr) (EII(Scr)) of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS), was determined. EllScr requires an EnzymeIII, the product of the gene crr, for full activity. The gene scrA is preceded immediately by a classical Shine-Dalgarno sequence (AAGAGGGTA). It contains 1368 nucleotides with an increased GC-content (58%) corresponding to a polypeptide of 455 amino acid residues (Mr 47,500). The protein has the hydropathic profile (average hydropathy +0.82) of an integral membrane protein lacking extended alpha-helical structures and a signal peptide. Comparison with the sequence of the beta-glucoside-specific EnzymeII (EII(Bgl), 625 amino acids, Mr 66,480; Bramley and Kornberg, 1987a; Schnetz et al., 1987) revealed strong homologies between EiI(Scr) and the first 458 residues of EII(Bgl). The 162 carboxyterminal residues of EII(Bgl), however, showed a high homology with the sequence of EnzymeIII (Nelson et al., 1984), a homology also described recently by Bramley and Kornberg (1987b). The evolutionary and functional significance of the similarities with four other EnzymesII is discussed.

Amino Acid Sequence↗