The effect of pyridoxal phosphate on the tryptophanases of five species of Enterobacteriaceae]

Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris et P. morganii tryptophanases (TPases) were studied for the spectral forms of the enzyme. The pH effect on the absorption spectrum and on the enzyme specific activity revealed that the coli group TPases are identical with but differ from Proteus TPases which differ themselves. The coli group TPases attach 4 mol of pyridoxal phosphate (PLP)/mol of enzyme, independently of the pH in the presence of K(plus) ions, and 9 mol of PLP/mol of enzyme must be reduced to achieve complete inactivation. The Proteus TPases attach 4 mol of PLP/mol of enzyme at PH 6.8, and 3 mol of PLP/mol of enzyme at pH 7.8 in K(plus) buffer. In P. morganii, 7 mol of PLP/mol of enzyme must be reduced to inactivate the enzyme, whereas P. vulgaris TPase cannot be completely inactivated by this method. These five TPases attach only 3 mol of PLP/mol of enzyme in a Na(plus) buffer, independently of the pH.     

Structure of the O-specific polysaccharide of Proteus vulgaris O37 and close serological relatedness of the lipopolysaccharides of P. vulgaris O37 and P. vulgaris O46

The O-specific polysaccharide (O-antigen) of the lipopolysaccharide (LPS) of Proteus vulgaris O37 was studied by (1)H and (13)C nuclear magnetic resonance spectroscopy before and after O-deacetylation and found to be structurally similar to that of P. vulgaris O46 studied earlier. The two polysaccharides have the same carbohydrate backbone and differ in the position and number of the O-acetyl groups only. Studies with O-antisera against the two strains using passive hemolysis test, enzyme immunosorbent assay, and Western blot revealed close serological relatedness of the LPSs of P. vulgaris O37 and O46. The O-acetyl groups were found to be of little importance for manifesting the O-specificity but to interfere with binding of anti-P. vulgaris O37 serum to P. vulgaris O46 antigen. Based on the data obtained, it was proposed to combine the strains studied in one Proteus serogroup O37 as subgroups O37a,37b and O37a,37c. A cross-reactivity of O-antisera against P. vulgaris O37 and O46 was observed with LPSs of three more Proteus strains, which could be substantiated by the presence of a common disaccharide fragment in the O-antigens.     

Structure of the glycerol phosphate-containing O-polysaccharides and serological studies of the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 classified into a new Proteus serogroup, O54

O-Polysaccharides were obtained from the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 and studied by chemical analyses and one- and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy, including rotating-frame nuclear Overhauser effect spectroscopy, H-detected (1)H,(13)C heteronuclear single-quantum spectroscopy and (1)H,(31)P heteronuclear multiple-quantum spectroscopy experiments. The Proteus mirabilis OE polysaccharide was found to have a trisaccharide repeating unit with a lateral glycerol phosphate group. The Proteus vulgaris TG 103 produces a similar O-polysaccharide, which differs in incomplete substitution with glycerol phosphate (c. 50% of the stoichiometric amount) and the presence of an O-acetyl group at position 6 of the 2-acetamido-2-deoxygalactose (GalNAc) residue. These structures are unique among the known bacterial polysaccharide structures. Based on the structural and serological data of the lipopolysaccharides, it is proposed to classify both strains studied into a new Proteus serogroup, O54, as two subgroups, O54a,54b and O54a,54c. The serological relatedness of the Proteus O54 and some other Proteus lipopolysaccharides is discussed.     

Sigmoid kinetics of human erythrocyte glucose-6-phosphate dehydrogenase

Several disagreements and inconsistencies have appeared regarding whether human erythrocyte glucose-6-phosphate dehydrogenase exhibits sigmoid or classical kinetics with respect to NADP+ binding. The latest report is that the purified enzyme exhibits classical kinetics while the intracellular enzyme exhibits sigmoid kinetics (H. N. Kirkman, and G. F. Gaetani (1986) J. Biol. Chem. 261, 4033-4038). The various investigations were carried out at fixed pH, ionic strength, and temperature. The steady-state kinetics of crude and purified erythrocyte glucose-6-phosphate dehydrogenase are reported here at various temperatures, ionic strengths, and pH values and as a function of glucose 6-phosphate concentration. Sigmoid kinetics were observed for both purified and crude enzyme samples at high pH, temperature, ionic strength, and concentration of glucose 6-phosphate with Hill coefficients varying between 1.40 and 1.90. In contrast, at low pH, temperature, and ionic strength, the crude enzyme samples exhibit sigmoid kinetics while the purified samples exhibit classical kinetics despite the high concentration of glucose 6-phosphate. High concentrations of glucose 6-phosphate and factors favoring the enzyme in the dimeric form are necessary conditions for the observation of sigmoid kinetics in human erythrocyte glucose-6-phosphate dehydrogenase. These factors are high pH, ionic strength, and temperature. The observed sigmoid kinetics in this enzyme is explained as arising from tetramer-dimer transitions.     

Depolymerization of chondroitin C Sulfate by immobilized Proteus vulgaris cells

Chondroitinase ABC catalyzing the depolymerization of chondroitin sulfate was induced by incubating the Proteus vulgaris cells in a medium containing chondroitin C sulfate as an inducer. Incubation of P. vulgaris cells for 12 h in the presence of 0.3% inducer was optimal to obtain the cells with highly active chondroitinase ABC. Such cells were immobilized in k-carrageenan gel lattice, and some properties of chondroitinase ABC in immobilized cells were studied in comparison with those of the enzyme without immobilization (free enzyme). The stabilities of the enzyme toward heat and storage were remarkably improved by immobilizing the cells in k-carrageenan gel lattice. Optimal pH and temperature for activity of the enzyme were slightly shifted to the alkaline region and higher temperature by immobilization and were 9.0 and 35 degrees C, respectively.     

Isolation and various characteristics of tryptophan deaminase from Proteus vulgaris

Tryptophan deaminase was isolated from Proteus vulgaris and purified. The procedure for enzyme purification included the cell destruction on USD-1, fractionation by ammonium sulphate, gel chromatography on ultragel AcA34, ion exchange chromatography on DEAE-cellulose. A degree of the enzyme purification--95, yield--5.7%. The pH optimum was 7.5, the temperature optimum--47 degrees C. The enzyme molecular weight (105 kD) was estimated by gel chromatography on Sephadex G-200, Km--5.0 mM in the K-phosphate buffer (pH 7.5). The SH groups are supposed to be present in the active site of the enzyme. The enzyme does not accelerate oxidation deamination of phenylalanine and tyrosine.     

Phospholipase D activity of gram-negative bacteria.

A phospholipase hydrolyzing cardiolipin to phosphatidic acid and phosphatidyl glycerol was characterized in gram-negative bacteria but was absent in preparations of gram-positive bacteria, Saccharomyces cerevisiae, and rat liver mitochondria. In cell-free extracts of Escherichia coli, Salmonella typhimurium, Proteus vulgaris, and Pseudomonase aeruginosa, this cardiolipin-hydrolyzing enzyme had similar pH and Mg2+ requirements and displayed a specificity which excluded phosphatidyl glycerol and phosphatidyl ethanolamine as substrates.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition

The kinetic mechanisms of the NAD- and NADP-linked reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides were examined using product inhibition, dead-end inhibition and alternate substrate experiments. The results are consistent with a steady-state random mechanism for the NAD-linked and an ordered, sequential mechanism with NADP+ binding first for the NADP-linked reaction. Thus, the enzyme can bind NADP+, NAD+, and glucose 6-phosphate, but the enzyme-glucose 6-phosphate complex can react only with NAD+, not with NADP+. This affects the rate equation for the NADP-linked reaction by introducing a term for a dead-end enzyme-glucose 6-phosphate complex. The kinetic mechanisms represent revisions of those proposed previously (C. Olive, M.E. Geroch, and H.R. Levy, 1971, J. Biol. Chem. 246, 2047-2057) and provide a kinetic basis for the regulation of coenzyme utilization of the enzyme by glucose 6-phosphate concentration (H.R. Levy, and G.H. Daouk, 1979, J. Biol. Chem. 254, 4843-4847) and NADPH/NADP+ concentration ratios (H.R. Levy, G.H. Daouk, and M.A. Katopes, 1979, Arch, Biochem. Biophys. 198, 406-413). The kinetic mechanisms were found to be the same at pH 6.2 and pH 7.8. The kinetics of ATP inhibition of the NAD- and NADP-linked reactions were examined at pH 6.2 and pH 7.8. The results are interpreted in terms of ATP addition to binary enzyme-coenzyme and enzyme-glucose 6-phosphate complexes.     

Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups

An acidic branched O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Proteus genomospecies 4 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected 1H, 13C HSQC experiments. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established, which is unique among Proteus polysaccharide structures: [structure: see text] where Qui3NAc stands for 3-acetamido-3,6-dideoxyglucose. Based on the O-polysaccharide structure and serological data, we propose classifying Proteus genomospecies 4 into a new, separate Proteus serogroup, O56. A weak cross-reactivity of Proteus genomospecies 4 antiserum with LPS of Providencia stuartii O18 and Proteus vulgaris OX2 was observed and is discussed in view of a similarity of the O-polysaccharide structures. Structural and serological investigations showed that Proteus genomospecies 5 and 6 should be classified into the existing Proteus serogroups O8 and O69, respectively.     

Structure of the O-specific polysaccharide of Proteus vulgaris O45 containing 3-acetamido-3,6-dideoxy-D-galactose

An O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O45 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established:-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Fucp3NAc4Ac-(1-->where Fuc3NAc4Ac is 3-acetamido-4-O-acetyl-3,6-dideoxygalactose. A cross-reactivity of anti-P. vulgaris O45 serum was observed with several other Proteus lipopolysaccharides, which contains Fuc3N derivatives.     

Structure of the O-polysaccharide of Proteus vulgaris O44: a new O-antigen that contains an amide of D-glucuronic acid with L-alanine

The O-polysaccharide of Proteus vulgaris O44, strain PrK 67/57 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H, 13C HMQC, HMQC-TOCSY and HMBC experiments. The polysaccharide was found to contain an amide of D-glucuronic acid with L-alanine [D-GlcA6(L-Ala)], and the following structure of the linear pentasaccharide repeating unit was established: [structure: see text]. The structural data of the O-polysaccharide and the results of serological studies with P. vulgaris O44 O-antiserum showed that the strain studied is unique among Proteus bacteria, which is in agreement with its classification in a separate Proteus serogroup, O44.     

Characterization of recombinant Aspergillus fumigatus mannitol-1-phosphate 5-dehydrogenase and its application for the stereoselective synthesis of protio and deuterio forms of D-mannitol 1-phosphate

A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a approximately 40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 degrees C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing (4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol 1-phosphate at pH 7.1 and 25 degrees C revealed a primary kinetic isotope effect of 2.9+/-0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions.     

Antagonistic interrelationships of Bifidobacterium bifidum i Proteus vulgaris in vitro in the digestive tract of gnotobiotic chicks]

The antagonistic relations between Bacterium bifidum, strain I/850 phi, and Proteus vulgaris, strain F-30, were studied. These organisms, when introduced together in equal doses into the digestive tract of gnotobiotic chickens in a single administration, were shown to create certain ecological correlations in various organs with the prevalence of bifidobacteria which exerted no negative influence on Proteus vulgaris. The additional daily administration of bifidobacteria for 3 days running in doses 1000 times as great as the initial dose, the content of both dibifobacteria and Proteus vulgaris in the intestine being at that time at its maximum, resulted in the suppression of the growth of Proteus vulgaris. Our findings indicate that the influence of the pH of the medium should be considered in order to obtain the evidence of significantly pronounced antagonistic relations between the two organisms in vitro.     

Structure of the O-polysaccharide leads to classification of Proteus penneri 31 in Proteus serogroup O19

O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C and 1H,31P HMQC experiments, demonstrated the following structure of the polysaccharide: [carbohydrate structure: see text] where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and alpha-L-FucNAc-(1-->3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31.     

Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris.

Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography.     

Structure of the N-acetyl-L-rhamnosamine-containing O-polysaccharide of Proteus vulgaris TG 155 from a new Proteus serogroup, O55

The O-polysaccharide of the lipopolysaccharide (LPS) of Proteus vulgaris TG 155 was found to contain 2-acetamido-2,6-dideoxy-L-mannose (N-acetyl-L-rhamnosamine, L-RhaNAc), a monosaccharide that occurs rarely in Nature. The following structure of the O-polysaccharide was established by NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H,13C HSQC experiments, along with chemical methods: [carbohydrate structure in text] Rabbit polyclonal O-antiserum against P. vulgaris TG 155 reacted with both core and O-polysaccharide moieties of the homologous LPS but showed no cross-reactivity with other LPS from the complete set of serologically different Proteus strains. Based on the unique O-polysaccharide structure and the serological data, we propose classifying P. vulgaris TG 155 into a new, separate Proteus O-serogroup, O55.     

Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris

Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography.     

Purification and properties of aldehyde dehydrogenase from Proteus vulgaris.

NADP-linked aldehyde dehydrogenase (aldehyde : NADP+ oxidoreductase, EC 1.2.1.4) was purified from Proteus vulgaris to the stage of homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 130000 by gel filtration. The enzyme which was crystallized from ammonium sulfate solution, lost its activity. The enzyme did not require coenzyme A, and the reaction was completely dependent on ammonium ions which could be partially replaced by Rb+ or K+. The optimum pH was about 9. Broad substrate specificity was observed and Km values for propionaldehyde, acetaldehyde and isovaleraldehyde were 1.7 - 10(-5), 4 - 10(-5) and 3 - 10(-5) M, respectively. The physiological role of the enzyme in living cells is obscure, but might account for another degradative pathway of L-leucine in P. vulgaris differing from the established pathway.     

Purification and properties of NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum)

The NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (formerly known as Acetobacter xylinum) has been purified to apparent homogeneity. The sequence of the 10 N-terminal amino acids was determined. The subunit molecular weight of the enzyme is 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gel filtration studies under nondenaturing conditions revealed that the molecular weight of the enzyme is 200,000 to 220,000 at pH 6.5 and 9.5, suggesting that the native enzyme is a tetramer. Specificity studies at both pH 6.5 and 9.5 demonstrated that the enzyme is a typical NADP-preferring glucose-6-phosphate dehydrogenase. The enzyme's catalytic activity increases with increasing pH, kcat being approximately 4 times greater at pH 9.5 than at pH 6.7 and the Km for NADP+ being 3 times lower at the higher pH; but the Km for glucose 6-phosphate is nearly 20 times higher at pH 9.5 than at pH 6.7, suggesting that the enzyme is catalytically more efficient at the lower pH. At pH 6.7, initial velocity measurements, product inhibition by NADPH, and inhibition by glucosamine 6-phosphate yielded results that were consistent with a steady-state random mechanism. At pH 9.5, steady-state kinetic analyses suggested that the mechanism is ordered, with coenzyme binding first, but nonlinear double-reciprocal plots were observed in the presence of NADPH when glucose 6-phosphate was varied and a complete kinetic analysis was not undertaken. Among several nucleotides and potential inhibitory ligands examined, only 2',5'-ADP inhibited the enzyme significantly.     

Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

After removal of tightly bound NAD(+) by using charcoal, a preparation of d-glucose 6-phosphate-1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-(3)H]NADH to give [5-(3)H]-glucitol 6-phosphate and [5-(3)H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [(3)H]myoinositol 1-phosphate in the presence of [4-(3)H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH(4) in the presence of NAD(+). Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD(+). There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15x10(5).