Cloning of the Escherichia coli sor genes for L-sorbose transport and metabolism and physical mapping of the genes near metH and iclR.

The sor genes for L-sorbose (Sor) degradation of Escherichia coli EC3132, a wild-type strain, have been cloned on a 10.8-kbp fragment together with parts of the metH gene. The genes were mapped by restriction analysis, by deletion mapping, and by insertion mutagenesis with Tn1725. Seven sor genes with their corresponding gene products have been identified. They form an operon (gene order sorCpCDFBAME) inducible by L-sorbose, and their products have the following functions: SorC (36 kDa), regulatory protein with repressor-activator functions; SorD (29 kDa), D-glucitol-6-phosphate dehydrogenase; SorF and SorB (14 and 19 kDa, respectively), and SorA and SorM (27 and 29 kDa, respectively), two soluble and two membrane-bound proteins, respectively, of an L-sorbose phosphotransferase transport system; SorE (45 kDa), sorbose-1-phosphate reductase. The sor operon from E. coli EC3132 thus is identical to the operon from Klebsiella pneumoniae KAY2026. On the basis of restriction mapping followed by Southern hybridization experiments, the sor genes were mapped at 91.2 min on the chromosome, 3.3 kbp downstream of the metH-iclR gene cluster, and shown to be transcribed in a counterclockwise direction. The chromosomal map of the Sor+ strain EC3132 differs from that of the Sor- strain K-12 in approximately 8.6 kbp.     

Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli.

D-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and D-glucitol-6-phosphate dehydrogenase (EC 1.1.1.140) were purified to apparent homogeneity in good yields from Escherichia coli. The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined. Both enzymes showed absolute specificities for their substrates. The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively. It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer. These two proteins differed in several fundamental respects.     

Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction.

Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6-phosphate dehydrogenase gene. Lys-21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate-5'-adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6-phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558-5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys-21. Both mutant enzymes showed increased Km-values for glucose 6-phosphate compared to wild-type enzyme: 1.4-fold (NAD-linked reaction) and 2.1-fold (NADP-linked reaction) for the K21R enzyme, and 36-fold (NAD-linked reaction) and 53-fold (NADP-linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5- and 3.2-fold, compared to the wild-type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat for the NAD- and NADP-linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD-linked reaction by 26% and decreased by 30% in the NADP-linked reaction from the values for the wild-type enzyme. The data are consistent with Lys-21 participating in the binding of the phosphate group of the substrate to the enzyme via charge-charge interaction.     

Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria

Sucrose-positive derivatives of Escherichia coli K-12, containing the plasmid pUR400, and of Klebsiella pneumoniae hydrolyse intracellular sucrose 6-phosphate by means of an invertase into D-glucose 6-phosphate and free D-fructose. The latter is phosphorylated by an ATP-dependent fructokinase (gene scrK of an scr regulon) to D-fructose 6-phosphate. The lack of ScrK does not cause any visible phenotype in wild-type strains of both organisms. Using genes and enzymes normally involved in D-arabinitol metabolism from E. coli C and K. pneumoniae, derivatives of E. coli K-12 were constructed which allowed the identification of scrK mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose plasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases--the equivalent of a peptide containing 307 amino acid residues (Mr 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginolyticus (57%) also involved in sucrose metabolism, lower overall identity (39%) to a D-ribose-kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP-binding sites.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Expression and purification of active human internal His(6)-tagged L-glutamine: D-Fructose-6P amidotransferase I

Human L-glutamine: D-fructose-6-phosphate amidotransferase (Gfat1), a recognized target in type 2 diabetes complications, was expressed in Sf9 insect cells with an internal His(6)-tag and purified to homogenity. Two different microplate assays that quantify, respectively D-glucosamine-6-phosphate and L-glutamate were used to analyze the enzyme kinetic properties. The recombinant human L-glutamine: D-fructose-6-phosphate amidotransferase isoform 1 exhibits Michaelis parameters K(m)(Fru-6P)=0.98 mM and K(m)(Gln)=0.84 mM which are similar to the values reported for the same enzyme from different sources. The stimulation of hydrolysis of the alternate substrate L-glutamine para-nitroanilide by D-fructose-6P (Fru-6P) afforded a K(d) of 5 microM for Fru-6P.     

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.     

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases of Escherichia coli C and Klebsiella pneumoniae M5a1 show very high N-terminal sequence homology

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) dehydrogenase from Escherichia coli C and Klebsiella pneumoniae M5a1 have been purified and some of their properties studied. The apparent Km values for NAD and CHMS were 11.7 +/- 1.5 microM and 5.2 +/- 1.9 microM, respectively, for the K. pneumoniae enzyme, and 19.5 +/- 2.7 microM and 9.2 +/- 1.4 microM, respectively, for the E. coli enzyme. Both enzymes were optimally active at pH 7.5 in sodium phosphate buffer. They had subunit molecular weights of 52,000 (+/- 1000) and the native enzymes appeared to be dimers of identical subunits. The first 20 residues of their N-terminal amino acid sequences were 90% homologous. A degenerate oligonucleotide probe constructed to a six amino acid sequence common to both enzymes gave strong hybridization with DNA from E. coli strains B and W as well as with E. coli C and K. pneumoniae but little or no hybridization to DNA from E. coli K12 or Pseudomonas putida.     

Two mammalian glucosamine-6-phosphate deaminases: a structural and genetic study

Glucosamine-6-phosphate deaminase (EC 3.5.99.6) is an allosteric enzyme that catalyzes the reversible conversion of D-glucosamine-6-phosphate into D-fructose-6-phosphate and ammonium. Here we describe the existence of two mammalian glucosamine-6-phosphate deaminase enzymes. We present the crystallographic structure of one of them, the long human glucosamine-6-phosphate deaminase, at 1.75 A resolution. Crystals belong to the space group P2(1)2(1)2(1) and present a whole hexamer in the asymmetric unit. The active-site lid (residues 162-182) presented significant structural differences among monomers. Interestingly the region with the largest differences, when compared with the Escherichia coli homologue, was found to be close to the active site. These structural differences can be related to the kinetic and allosteric properties of both mammalian enzymes.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Purification and characterization of glutamine:fructose 6-phosphate amidotransferase from rat liver

The enzyme glutamine:fructose 6-phosphate amidotransferase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The K(M) values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4, 4'-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the K(inact) of 5.0 microM. Kinetic studies also indicated that 4,4'-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801-810).     

Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes.

Enzyme IIIMtl is part of the mannitol phosphotransferase system of Enterococcus faecalis. It is phosphorylated in a reaction sequence requiring enzyme I and heat-stable phosphocarrier protein (HPr). The phospho group is transferred from enzyme IIIMtl to enzyme IIMtl, which then catalyzes the uptake and concomitant phosphorylation of mannitol. The internalized mannitol-1-phosphate is oxidized to fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. In this report we describe the cloning of the mtlF and mtlD genes, encoding enzyme IIIMtl and mannitol-1-phosphate dehydrogenase of E. faecalis, by a complementation system designed for cloning of gram-positive phosphotransferase system genes. The complete nucleotide sequences of mtlF, mtlD, and flanking regions were determined. From the gene sequences, the primary translation products are deduced to consist of 145 amino acids (enzyme IIIMtl) and 374 amino acids (mannitol-1-phosphate dehydrogenase). Amino acid sequence comparison confirmed a 41% similarity of E. faecalis enzyme IIIMtl to the hydrophilic enzyme IIIMtl-like portion of enzyme IIMtl of Escherichia coli and 45% similarity to enzyme IIIMtl of Staphylococcus carnosus. The putative N-terminal NAD+ binding domain of mannitol-1-phosphate dehydrogenase of E. faecalis shows a high degree of similarity with the N terminus of E. coli mannitol-1-phosphate dehydrogenase (T. Davis, M. Yamada, M. Elgort, and M. H. Saier, Jr., Mol. Microbiol. 2:405-412, 1988) and the N-terminal part of the translation product of S. carnosus mtlD, which was also determined in this study. There is 40% similarity between the dehydrogenases of E. faecalis and E. coli over the whole length of the enzymes. The organization of mannitol-specific genes in E. faecalis seems to be similar to the organization in S. carnosus. The open reading frame for enzyme IIIMtl E. faecalis is followed by a stem-loop structure, analogous to a typical Rho-independent terminator. We conclude that the mannitol-specific genes are organized in an operon and that the gene order is mtlA orfX mtlF mtlD.     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.     

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor?manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor?manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor?manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.     

Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae.

The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.