Purification and characterization of the bifunctional uridylyltransferase and the signal transducing proteins GlnB and GlnK from Herbaspirillum seropedicae.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme that has a central role in the general nitrogen regulatory system NTR. In enterobacteria, GlnD uridylylates the PII proteins GlnB and GlnK under low levels of fixed nitrogen or ammonium. Under high ammonium levels, GlnD removes UMP from these proteins (deuridylylation). The PII proteins are signal transduction elements that integrate the signals of nitrogen, carbon and energy, and transduce this information to proteins involved in nitrogen metabolism. In Herbaspirillum seropedicae, an endophytic diazotroph isolated from grasses, several genes coding for proteins involved in nitrogen metabolism have been identified and cloned, including glnB, glnK and glnD. In this work, the GlnB, GlnK and GlnD proteins of H. seropedicae were overexpressed in their native forms, purified and used to reconstitute the uridylylation system in vitro. The results show that H. seropedicae GlnD uridylylates GlnB and GlnK trimers producing the forms PII (UMP)(1), PII (UMP)(2) and PII (UMP)(3), in a reaction that requires 2-oxoglutarate and ATP, and is inhibited by glutamine. The quantification of these PII forms indicates that GlnB was more efficiently uridylylated than GlnK in the system used.

TbMP57 is a 3' terminal uridylyl transferase (TUTase) of the Trypanosoma brucei editosome.

RNA editing produces mature trypanosome mitochondrial mRNAs by uridylate (U) insertion and deletion. In insertion editing, Us are added to the pre-mRNA by a 3' terminal uridylyl transferase (TUTase) activity. We report the identification of a TUTase activity that copurifies with in vitro editing and is catalyzed by the integral editosome protein TbMP57. TbMP57 catalyzes the addition of primarily a single U to single-stranded (ss) RNA and adds the number of Us specified by a guide RNA to insertion editing-like substrates. TbMP57 is distinct from a previously identified TUTase that adds many Us to ssRNA and which we find is neither a stable editosome component nor does it add Us to editing-like substrates. Recombinant TbMP57 specifically interacts with the editosome protein TbMP81, and this interaction enhances the TUTase activity. These results suggest that TbMP57 catalyzes U addition to pre-mRNA during editing.

Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues.

SETTING: Mycobacterial galactofuran is essential to the linking of the peptidoglycan and mycolic acid cell wall layers. Galactofuran biosynthesis should thus be essential for viability. OBJECTIVE: The objective was to determine the pathway of galactofuranosyl biosynthesis and to clone a gene encoding an essential enzyme necessary for its formation. DESIGN: Specific enzymatic conversions involved in formation of galactopyranose and galactofuranose residues in other bacteria were tested for in Mycobacterium smegmatis. M. tuberculosis deoxyribonucleic acid (DNA) was identified by homology. RESULTS: It was shown that the de novo synthesis of the galactose carbon skeleton occurred in M. smegmatis by the transformation of UDP-glucopyranose to UDP-galactopyranose via the enzyme UDP-glucose 4-epimerase (E.C. 5.1.3.2). The N-terminal sequence of this enzyme was obtained after purification. The galactose salvage pathway enzyme, UDP-glucose-galactose-1-phosphate uridylyltransferase (E.C. 2.7.7.12), was also shown to be present. The critical biosynthetic transformation of the galactopyranose to galactofuranose ring form was shown to occur at the sugar nucleotide level via the enzyme UDP-galactopyranose mutase (E.C. 5.4.99.9). The M. tuberculosis DNA encoding this enzyme was sequenced, the gene expressed in Escherichia coli, and the expected enzymatic activity demonstrated. CONCLUSION: Galactofuranose biosynthesis can now be pursued as a potential drug target in M. tuberculosis.

Purification and characterization of a galactose-1-phosphate: UDP-glucose uridyltransferase from the red alga Galdieria sulphuraria.

The galactose-1-phosphate uridyltransferase of the red alga Galdieria sulphuraria has been purified about 1800-fold to a final specific activity of approximately 140 U/mg protein. The purification involved chromatography on DEAE-Fractogel, hydroxyapatite, decyl-agarose, and DEAE-Tentacle gel. After SDS/PAGE, the enzyme preparation showed only one protein band of 42 kDa. The enzyme is a homodimer with a molecular mass of 82 kDa as estimated from the sedimentation velocity or 60 kDa as estimated by gel filtration. It has a broad pH optimum between pH 7 and pH 9. The apparent Km values for the forward and backward reactions are Km(Glc1P) = 105 microM, Km(UDP-galactose) = 30 microM, Km(Gal1P) = 400 microM, and Km(UDP-Glc) = 20 microM. The activation energy of the reaction is 45 kJ mol-1. The enzyme is specific for the galactose 1-phosphate to UDP-galactose interconversion in the Leloir pathway while the alternate enzyme for the Isselbacher pathway, UDP-galactose pyrophosphorylase, could not be detected in G. sulphuraria.

Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII uridylyltransferase and uridylyl-removing enzyme.

Uridylyltransferase, a component of the covalent modification cascade system that controls glutamine synthetase activity in Escherichia coli, has been purified to apparent homogeneity. The purification was facilitated by the use of an E. coli strain which carries multiple copies of a ColE1-hybrid plasmid containing the glnD gene that encodes uridylyltransferase and which overproduces its synthesis by 25-fold. Gel electrophoresis and high pressure liquid chromatography studies show that the native enzyme is a single polypeptide chain of Mr = 95,000 +/- 5,000. The purified enzyme catalyzes the uridylylation as well as the deuridylylation of the regulatory protein PII, demonstrating that a single bifunctional enzyme is involved in the covalent interconversion of PII. Gel filtration studies indicate that the enzyme undergoes slow irreversible aggregation during most steps of purification with a concomitant loss of activity.

Separation and characterization of two UTP-utilizing hexose phosphate uridylyltransferases from Entamoeba histolytica.

Two UTP-utilizing uridylyltransferases which react with both glucose 1-phosphate and galactose 1-phosphate were isolated from cell-free extracts of Entamoeba histolytica. The more specific of these enzymes, glucose-1-phosphate uridylyltransferase, acts preferentially on glucose 1-phosphate, having a maximum velocity 20-fold greater with this substrate than with galactose 1-phosphate. It was purified 200 fold with a 25% yield and has a molecular weight of 45 000. This enzyme requires a reducing agent for stability. The less specific transferase reacts with both hexose phosphates, having a maximum velocity of 1.35 times greater with galactose 1-phosphate. It was purified 1000 fold with a 20% yield, and has a molecular weight of 40 000. The common Leloir enzyme, UDP glucose-hexose-1-phosphate uridylytransferase (EC 2.7.7.12), was not found in this organism. To avoid confusion with the Leloir enzyme our experience suggests that the less specific enzyme, which is presently referred to in the literature as galactose-1-phosphate uridylyltransferase (EC 2.7.7.10), should be named UTP:hexose-1-phosphate uridylyltransferase (EC 2.7.7.?). The more specific enzyme (EC 2.7.7.9) should be more clearly named UTP:glucose-1-phosphate uridylyltransferase.

Purification of human liver uridylyl transferase and comparison with the erythrocyte enzyme.

Uridylyl transferase (UDP glucose: alpha-D-galactose 1 phosphate uridylyl transferase, EC 2.7.7.12) has been purified 1350-fold from human liver to complete homogeneity. The purification procedure involved ammonium sulfate fractionation, batch treatment, chromatography on DEAE-cellulose, hexylagarose and hydroxylapatite. The specific activity of the homogeneous enzyme was 27 units/mg protein. The liver enzyme was compared to the red cell enzyme previously purified by us. The liver enzyme was similar to the red cell enzyme with respect to subunit molecular weight, kinetic studies and immunological properties. Differences in electrophoretic behaviour were found: the liver transferase has a more basic pI (between 5.5 and 5.8) than that of the erythrocyte enzyme (pI between 5.0 and 5.45). It is very likely that the liver uridylyl transferase and the red blood cell transferase are the same enzymes with post-translational modifications.

Purification and characterization of human erythrocyte uridylyl transferase.

A new method for the purification of human erythrocyte uridylyl transferase (UDPglucose: alpha-D-galactose-1-phosphate uridylyltransferase EC 2.7.7.12) is described. It consists of a hydrophobic purification step associated with hydroxyapatite chromatography and provided for the first time a purification of more than 45 000-fold with a high activity (15 I.U/mg) and a yield of 32%. We show that the enzyme is a dimer and has a molecular weight of 88 000. It can be resolved into three bands by isoelectric focusing with an apparent pI between 5.0 and 5.4. It could be shown by steady-state initial rate measurements that the interconversion of the two substrates of human transferase (Gal-1-P and UDP-glucose) follows ping-pong bi-bi kinetics, with Km values of 0.2 and 0.065 mM, respectively.

Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate.

Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.

Enzymes of galactose utilization in the rat tapeworm, Hymenolepis diminuta.

1. Crude enzyme preparations from Hymenolepis diminuta contained galactokinase, galactose 1-phosphate uridyl transferase and UDPgalactose 4-epimerase activity, although their specific activities were low. 2. Galactose 1-phosphate non-competitively inhibited galactose phosphorylation. This inhibition, together with the low specific activities of the enzymes in the pathway of galactose utilization, probably accounts for the inadequacy of galactose as a main nutritive carbohydrate for development of the worm.