Mechanism of uridine production by Bacillus subtilis mutants

Summary Pyrimidine analogue-resistant mutants of Bacillus subtilis were found to produce a large amount of uridine. One of them accumulated 55 mg/ml of uridine in culture medium. The changes in enzymes involved in the metabolism of uridine 5-monophosphate (UMP) were examined with this mutant. All six enzymes of de novo UMP biosynthesis were completely free from regulation by uridine compounds, and the activities of these enzymes were 16- to 30-fold higher than those of the enzymes of the parental strain. In the mutant strain, the level of uridine phosphorylase, responsible for converting uridine to uracil, was extremely low, compared with that of the parental strain. No apparent change was observed between the strains in the activity of UMP dephosphorylation or uracil phosphoribosyltransferase. The implication of these findings is discussed in relation to the overproduction of uridine by the mutant.Microbial production of uridine. Part III     

Structural and functional investigations of Ureaplasma parvum UMP kinase--a potential antibacterial drug target

The crystal structure of uridine monophosphate kinase (UMP kinase, UMPK) from the opportunistic pathogen Ureaplasma parvum was determined and showed similar three-dimensional fold as other bacterial and archaeal UMPKs that all belong to the amino acid kinase family. Recombinant UpUMPK exhibited Michaelis-Menten kinetics with UMP, with K(m) and V(max) values of 214 +/- 4 microm and 262 +/- 24 micromol.min(-1).mg(-1), respectively, but with ATP as variable substrate the kinetic analysis showed positive cooperativity, with an n value of 1.5 +/- 0.1. The end-product UTP was a competitive inhibitor against UMP and a noncompetitive inhibitor towards ATP. Unlike UMPKs from other bacteria, which are activated by GTP, GTP had no detectable effect on UpUMPK activity. An attempt to create a GTP-activated enzyme was made using site-directed mutagenesis. The mutant enzyme F133N (F133 corresponds to the residue in Escherichia coli that is involved in GTP activation), with F133A as a control, were expressed, purified and characterized. Both enzymes exhibited negative cooperativity with UMP, and GTP had no effect on enzyme activity, demonstrating that F133 is involved in subunit interactions but apparently not in GTP activation. The physiological role of UpUMPK in bacterial nucleic acid synthesis and its potential as target for development of antimicrobial agents are discussed.     

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.     

Coordinate overproduction of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase in hamster cells resistant to pyrazofurin and 6-azauridine.

Cells resistant to pyrazofurin and 6-azauridine have been selected from a simian virus 40-transformed Syrian hamster line and from a Chinese hamster lung line. By increasing the concentrations of inhibitors in several steps, mutant cells from both lines have been obtained which resist high concentrations (1 to 5 mM) of the two inhibitors separately or together. Orotidine-5'-phosphate decarboxylase (EC 4.1.1.23), the sixth and last enzyme in UMP biosynthesis, is inhibited by the nucleoside monophosphates derived from pyrazofurin or 6-azauridine. The activity of this enzyme is increased in each resistant cell line tested. Furthermore, there is a parallel increase in each case in the activity of the fifth enzyme of the pathway, orotate phosphoribosyltransferase (EC 2.4.2.10), which is not inhibited by pyrazofurin or 6-azauridine monophosphates, and the amount of increase is up to 67 times the level found in wild type cells. In contrast, the activities of the first three enzymes of UMP biosynthesis remain essentially unchanged in the mutants. Resistant Chinese hamster cells remain sensitive to 5-fluorouridine; this indicates that uridine kinase, the enzyme necessary to convert 6-azauridine to the monophosphate, is still functional.     

Atomic Resolution Structure of the Orotidine 5′-Monophosphate Decarboxylase Product Complex Combined with Surface Plasmon Resonance Analysis: IMPLICATIONS FOR THE CATALYTIC MECHANISM*

Orotidine 5′-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of its substrate by 17 orders of magnitude. One argument brought forward against steric/electrostatic repulsion causing substrate distortion at the carboxylate substituent as part of the catalysis has been the weak binding affinity of the decarboxylated product (UMP). The crystal structure of the UMP complex of ODCase at atomic resolution (1.03 Å) shows steric competition between the product UMP and the side chain of a catalytic lysine residue. Surface plasmon resonance analysis indicates that UMP binds 5 orders of magnitude more tightly to a mutant in which the interfering side chain has been removed than to wild-type ODCase. These results explain the low affinity of UMP and counter a seemingly very strong argument against a contribution of substrate distortion to the catalytic reaction mechanism of ODCase.     

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.     

Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid

Summary In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase^– mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura ^– phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.     

UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.     

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Uridine 5′-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mm N-nitroso-N-methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-fluoroorotic acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-fluoroorotic acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5′-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5′-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin2-CAT gene remained unchanged by introduction of the UMP synthase mutations.     

Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of UTP. They are hexamers regulated by GTP (allosteric activator) and UTP (inhibitor). We describe here the 2.8 angstroms crystal structure of Escherichia coli UMP kinase bound to GTP. The GTP-binding site, situated at 15 angstroms from the UMP-binding site and at 24 angstroms from the ATP-binding site, is delineated by two contiguous dimers. The overall structure, as compared with those bound to UMP, UDP, or UTP, shows a rearrangement of its quaternary structure: GTP induces an 11 degrees opening of the UMP kinase dimer, resulting in a tighter dimer-dimer interaction. A nucleotide-free UMP kinase dimer has an intermediate opening. Superposition of our structure with that of archaeal UMP kinases, which are also hexamers, shows that a loop appears to hamper any GTP binding in archeal enzymes. This would explain the absence of activating effect of GTP on this group of UMP kinases. Among GTP-binding residues, the Asp-93 is the most conserved in bacterial UMP kinases. In the previously published structures of E. coli UMP kinase, this residue was shown to be involved in hydrogen bonds between the subunits of a dimer. Its substitution by an alanine decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition. This demonstrates that the previously described mutual exclusion of these two nucleotides is mediated by Asp-93.     

Proteomic analysis of curdlan-producing Agrobacterium sp. in response to pH downshift

During batch cultivation of Agrobacterium sp. ATCC 31750, proteome analysis in response to a pH downshift from 7.0 to 5.5 was carried out using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. When the pH of the exponentially growing Agrobacterium sp. culture was downshifted to pH 5.5, the synthesis level of 27 intracellular proteins showed significant changes in level over a prolonged period of time compared with the batch culture controlled at pH 7.0. In particular, the intracellular protein level of the beta-1,3-glucan synthase catalytic subunit, UTP-glucose-1-phosphate uridylyltransferase, and phosphoglucomutase, which are key metabolic enzymes in the curdlan biosynthesis pathway, were more than 10-, 3- and 17-times higher in the low pH culture. On the other hand, the level of orotidine5-phosphate decarboxylase (conversion of OMP to UMP) was significantly up-regulated after pH downshift. The accumulation of UMP may direct the metabolic flow towards the biosynthetic route of UTP, which is a key metabolic precursor for UDP-glucose. Therefore, it is possible that increase of cellular metabolic enzymes during pH downshift culture can enhance the metabolic flux of the biosynthesis of key precursor, such as UTP- and UDP-glucose, resulting in an increase in curdlan biosynthesis.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells.

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.     

Biochemical and genetic characterization of a carbamyl phosphate synthetase mutant of Escherichia coli K12.

An unusual Escherichia coli K12 mutant for carbamyl phosphate synthetase is described. The mutation was generated by bacteriophage MUI insertion and left a 5% residual activity of the enzyme using either ammonia or glutamine as donors. The mutation is recessive to the wild-type allele and maps at or near the pyrA gene, but the mutant requires only arginine and not uracil for growth. By a second block in the pyrB gene it was possible to shift the accumulated carbamyl phosphate to arginine biosynthesis. The Km values and the levels of ornithine activation and inhibition by UMP were normal in the mutant enzyme.     

Localization of the site for the nucleotide effectors of Escherichia coli carbamoyl phosphate synthetase using site-directed mutagenesis

Replacement by alanine of Ser-948, Thr-974 and Lys-954 of Escherichia coli carbamoyl phosphate synthetase (CPS) shows that these residues are involved in binding the allosteric inhibitor UMP and the activator IMP. The mutant CPSs are active in vivo and in vitro and exhibit normal activation by ornithine, but the modulation by both UMP and IMP is either lost or diminished. The results demonstrate that the sites for UMP and IMP overlap and that the activator ornithine binds elsewhere. Since the mutated residues were found in the crystal structure of CPS near a bound phosphate, Ser-948, Thr-974 and Lys-954 bind the phosphate moiety of UMP and IMP.     

Characterization of UMP synthase in dairy cattle heterozygous for a lethal recessive trait

UMP synthase was characterized biochemically in dairy cattle heterozygous for a deficiency of this enzyme. Both activities comprising this bifunctional enzyme are decreased, with OMP decarboxylase more affected than orotate phosphoribosyltransferase. Immunotitration of UMP synthase activity revealed the presence of the protein product of the mutant allele in the heterozygous animals. UMP synthases from normal and deficient cattle were not distinguished from one another by kinetic constants, responses to inhibitors, pH profiles, or thermal lability. It was concluded that the 50% reduction in enzyme activity in heterozygous cattle is the result of the presence of only half the normal level of catalytically active UMP synthase.     

Orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase exist as multienzyme complex in human malaria parasite Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, totally depends on de novo pyrimidine biosynthetic pathway. Orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzymes in the pathway catalyzing formation of uridine 5'-monophosphate (UMP), remain largely uncharacterized in the protozoan parasite. In this study, we achieved purification of OPRT and OMPDC to near homogeneity from P. falciparum cultivated in vitro. The OPRT and OMPDC activities were co-eluted in all chromatographic columns during purification, suggesting the purified proteins exist as a multienzyme complex with a molecular mass of 140+/-8 kDa and contain two subunits each of OPRT and OMPDC. Monomeric forms of OPRT and OMPDC had molecular masses of 32+/-3 and 38+/-3 kDa, respectively, in agreement with those of proteins predicted from P. falciparum genome database. Interestingly, kinetic parameters and inhibitory constants of both OPRT and OMPDC activities were found to be different to those of the bifunctional human red cell UMP synthase. Our evidence provides the first example of OPRT and OMPDC existing as a multienzyme complex.     

A unique catalytic and inhibitor-binding role for Lys93 of yeast orotidylate decarboxylase.

The presence of a proton-donating catalytic amino acid side chain in orotidylate decarboxylase (ODCase) was sought by site-directed mutagenesis. Replacement of yeast ODCase Lys93 with a cysteine resulted in a mutant protein (K93C) with no measurable activity, representing a decrease in activity by a factor of, at most, 2 x 10(-8) times the activity of the wild-type enzyme. Treatment of this mutant protein with 2-bromoethylamine, designed to append Cys93 to yield S-(2-aminoethyl)cysteine, restored activity by a factor of at least 5 x 10(5) over the untreated mutant protein. Activity could not be restored by treatment with other brominated reagents designed to replace the epsilon-amino of S-(2-aminoethyl)Cys93 with a different functional group. The overall architecture of the K93C protein was not significantly changed, as judged by the similar dimerization properties (in the absence of ligands) of the mutant enzyme compared to the wild-type enzyme. The binding affinity of the substrate orotidylate was not measurably changed by the mutation, indicating that Lys93 has an essential role in catalysis which is mechanistically distinguishable from substrate binding. Apparently the mutation removes an integral portion of the active site and does not drastically affect the structural or substrate binding properties. However, the affinities of the mutant protein for the competitive inhibitors 6-azauridylate (6-azaUMP) and UMP are significantly altered from the pattern seen with the wild-type enzyme. The K93C protein has an affinity for the neutral ligand UMP which is greater than that for the anionic 6-azaUMP, in clear contrast to the preference for 6-azaUMP displayed by the wild-type enzyme. Lys93 is apparently critical for catalysis of the substrate to product and for the binding of anionic inhibitors; the data are discussed in terms of previously existing models for transition-state analogue inhibitor binding and catalysis.