Deoxyribokinase from Salmonella typhimurium. Purification and properties

Deoxyribokinase, which catalyzes the ATP-dependent phosphorylation of 2-deoxy-d-ribose to 2-deoxy-d-ribose-5-P as the first step in the inducible fermentation pathway for this sugar in Salmonella typhimurium, was purified approximately 600-fold from deoxyribose-grown cells. Apparent K_m′s for 2-deoxy-d-ribose and ATP were 0.1 and 0.5 mm, respectively. The enzyme had an absolute requirement for divalent cations which was best satisfied by Mg²⁺. Optimal activity was obtained in the presence of 0.5 m NH₄⁺ or Cs⁺. Rb⁺ and K⁺ also stimulated enzyme activity whereas Na⁺ and Li⁺ inhibited. d-Ribose and 2-deoxy-d-ribitol could replace 2-deoxy-d-ribose as phosphoryl acceptor, and several ribo- and deoxyribonucleotides could replace ATP as phosphoryl donor. Molecular weight determinations gave values of 67,800 for the native enzyme and 33,500 for the dissociated enzyme, suggesting the probable existence of two subunits of similar size.     

Purification and properties of trimethylamine N-oxide reductase from aerobic photosynthetic bacterium Roseobacter denitrificans.

Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90,000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172,000. The midpoint redox potential of heme c was +200 mV at pH 7.5. Absorption maxima appeared at 418,524, and 554 nm in the reduced state and 410 nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase.     

Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties.

The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized. Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis-Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G-200 of the native enzymes revealed a molecular weight for both enzymes of 138000 plus or minus 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 plus or minus 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight. When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical. 6M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active. Both enzymes could be dissociated into subunits by p-chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1-phosphates, and mixtures of phosphate and purine bases.     

Purification of pseudouridylate synthetase I from Salmonella typhimurium.

Pseudouridylate synthetase from Salmonella typhimurium has been purified 1,000 fold and is about 90% pure. The enzyme has a molecular weight of 50,000 daltons. In the presence of tRNA there is a change in molecular weight from 50.000 to 100.000. This change does not seem to be due to the formation of a tRNA-enzyme complex but rather to a tRNA induced dimerization. Other properties of the enzyme are described.     

Purification and kinetic properties of pyruvate kinase isoenzymes of Salmonella typhimurium.

Two forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Salmonella typhimurium were purified to homogeneity from the same cultures by (NH4)2SO4 fractionation and gel filtration, anion-exchange and affinity chromatography. Mr values, subunit structure, amino acid composition and activity and stability conditions were determined for the two forms. Kinetic and regulatory properties of the two purified isoenzymes were studied.     

Purification and characterization of a tRNA methylase from Salmonella typhimurium.

A tRNA methylase, in which supK strains of Salmonella typhimurium are deficient, was purified from strain LT2 and characterized. Column chromatography of protein extracts from wild-type cells on phosphocellulose, diethylaminoethyl-Sephadex A-50, and hydroxlapatite resulted in an enzyme that was estimated to be about 50% pure. tRNA from S. typhimurium which had been incubated at pH 9.0 served as a substrate for this methylase. The enzyme has a molecular weight of about 50,000 as estimated by gel chromatography and by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The optimal assay conditions, as well as the kinetics and stability of the enzyme, were studied. As with other tRNA-methylating enzymes, S-adenosylhomocysteine is a potent inhibitor.     

Tricarboxylate-binding proteins of Salmonella typhimurium. Purification, crystallization, and physical properties

Citrate transport in Salmonella typhimurium involves inducible periplasmic components. Two forms of a tricarboxylate-binding protein, C1 and C2, were isolated, in high yield, from the periplasm of a cyclic AMP phosphodiesterase mutant. These immunologically cross-reactive Mr = 29,000 proteins were crystallized using ammonium sulfate. CD measurements indicated considerable secondary structure: 24% a helix, and 12% beta structure. The amino acid compositions of C1 and C2 were identical. The NH2-terminal sequence of C1 was determined; C2 was found to have a blocked NH2 terminus (pyroglutamate). C1 and C2 are products of the same gene (Somers, J. M., and Kay, W. W. (1983) Mol. Gen. Genet. 190, 20-26). C1 and C2 bound a variety of citrate analogues and organic acids, with a predominant specificity for tricarboxylates (citrate KD 1.4 X 10(-7) M), and both required a deprotonated central carboxyl group for binding. Citrate was not bound to C protein as either a salt or metal ion complex.