Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae

In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.     

Expression and sequence analysis of a cDNA encoding the orotidine-5'-monophosphate decarboxylase domain from Ehrlich ascites uridylate synthase

Orotidine-5'-monophosphate decarboxylase (OD-Case) catalyzes the conversion of orotidine 5'-monophosphate to UMP. In mammals, ODCase is present as part of a bifunctional protein which also contains orotate phosphoribosyltransferase; the preceding enzyme in the de novo UMP biosynthetic pathway. We have isolated a plasmid (pMEJ) which contains a cDNA for the ODCase domain of UMP synthase. Insertion of this sequence into an Escherichia coli expression vector (pUC12) has allowed for the expression of ODCase and not orotate phosphoribosyltransferase in E. coli. The molecular weight of the expressed protein is 26,000-27,300 from immunoblot analysis which corresponds closely to the molecular weight of the ODCase domain (28,500) isolated by tryptic digestion of UMP synthase. We have sequenced the cDNA insert of pMEJ and deduced the amino acid sequence. The molecular weight of the ODCase domain calculated from the amino acid sequence in 28,654. Comparison of the deduced amino acid sequence from pMEJ with that for yeast ODCase (a monofunctional protein) demonstrated that 52% of the amino acids were identical when the two sequences are compared. Furthermore, several stretches of the amino acid sequence have 80% or greater absolute homology.     

Purification and characterization of yeast orotidine 5'-monophosphate decarboxylase overexpressed from plasmid PGU2

Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.     

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.     

Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase

The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli.     

Substrate binding induces domain movements in orotidine 5'-monophosphate decarboxylase

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyses the decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate (UMP). We have earlier determined the structure of ODCase from Escherichia coli complexed with the inhibitor 1-(5'-phospho-beta-d-ribofuranosyl)barbituric acid (BMP); here we present the 2.5 A structure of the uncomplexed apo enzyme, determined from twinned crystals. A structural analysis and comparison of the two structures of the E. coli enzyme show that binding of the inhibitor is accompanied by significant domain movements of approximately 12 degrees around a hinge that crosses the active site. Hence, the ODCase dimer, which contains two active sites, may be divided in three domains: a central domain that is fixed, and two lids which independently move 12 degrees upon binding. Corresponding analyses, presented herein, of the two Saccharomyces cerevisiae ODCase structures (with and without BMP) and the Methanobacterium thermoautotrophicum ODCase structures (with and without 6-aza UMP) show very similar, but somewhat smaller domain movements. The domain movements seem to be initiated by the phosphoryl binding to the enzyme and can explain why the binding of the phosphoryl group is essential for the catalytic function.     

The Leishmania donovani UMP synthase is essential for promastigote viability and has an unusual tetrameric structure that exhibits substrate-controlled oligomerization

The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.     

A reexamination of the substrate utilization of 2-thioorotidine-5'-monophosphate by yeast orotidine-5'-monophosphate decarboxylase

A potential alternate substrate for orotidine-5'-monophosphate decarboxylase, 2- thio-orotidine-5'-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry 31, 12155-12161). Characterization of the product was confirmed by mass spectrometry, (31)P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously. Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10(-7) from the k(cat) for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 microM, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase.     

Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5'-monophosphate decarboxylase

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5'-monophosphate, the last step in the de novo synthesis of uridine 5'-monophosphate. ODCase is a very proficient enzyme [Radzicka, A., and Wolfenden, R. (1995) Science 267, 90-93], enhancing the reaction rate by a factor of 10(17). This proficiency has been enigmatic, since it is achieved without metal ions or cofactors. Here we present a 2.5 A resolution structure of ODCase complexed with the inhibitor 1-(5'-phospho-beta-D-ribofuranosyl)barbituric acid. It shows a closely packed dimer composed of two alpha/beta-barrels with two shared active sites. The orientation of the orotate moiety of the substrate is unambiguously deduced from the structure, and previously proposed catalytic mechanisms involving protonation of O2 or O4 can be ruled out. The proximity of the OMP carboxylate group with Asp71 appears to be instrumental for the decarboxylation of OMP, either through charge repulsion or through the formation of a very short O.H.O hydrogen bond between the two carboxylate groups.     

The purification and preliminary characterization of UMP synthase from human placenta

Orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23) are the final two of six enzymatic steps required in the de novo biosynthesis of uridine 5'-monophosphate (UMP). Earlier work of this laboratory showed that, in the mouse Ehrlich ascites carcinoma, both of these enzymatic activities were contained on the single multifunctional polypeptide chain, UMP synthase. We report here that the placenta provided an available human source for UMP synthase with 40-fold higher orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase specific activities than erythrocytes, a human source previously used by others. By using the placenta as a source of UMP synthase and by developing a novel purification procedure different from that used in the purification of UMP synthase from the Ehrlich ascites carcinoma (the only other homogeneous preparation of a mammalian UMP synthase), we achieved the purification of human UMP synthase to apparent homogeneity. This represents the first publication to homogeneity of UMP synthase from a human source as well as from a source other than malignant cell lines. Contrary to earlier reports human placental UMP synthase was found to be a multifunctional protein containing both enzymatic activities on a single polypeptide of 51,000 molecular weight. Preliminary characterization of the human placental UMP synthase showed it to be similar to UMP synthase from the Ehrlich ascites carcinoma in subunit molecular weight, native molecular weight, isozyme pattern (although not absolute pI values), pH optima of enzymatic activities, and kinetic constants for orotidine 5'-monophosphate (Km) and 6-azauridine 5'-monophosphate (Ki) at the decarboxylase site.     

Enzymes of de novo pyrimidine biosynthesis in Babesia rodhaini

The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5'-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent Ki of 7 microM. Dihydro-5-azaorotate inhibited the DHO-DHase (Ki, 16 microM) and 5-azaorotate (Ki, 21 microM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (Ki, 0.3 microM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (Ki, 120 microM) and the purine nucleotide, XMP (Ki, 95 microM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

Orotate phosphoribosyltransferase and orotidylate decarboxylase from Crithidia luciliae: subcellular location of the enzymes and a study of substrate channeling

Orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) have been found to be particulate in the kinetoplastid protozoan, Crithidia luciliae. Sucrose density centrifugation indicated that these two enzymes are associated with the glycosome, a microbody which appears to be unique to the Kinetoplastida and which contains many of the glycolytic enzymes. The particulate location of OPRTase and ODCase was considered to be favorable for channeling of orotidine-5'-monophosphate (OMP), the product of the first enzyme and substrate for the second. The degree of channeling was determined by double radioactively labeled experiments designed to determine the relative efficiency of endogenous and exogenous OMP as substrates of ODCase. The efficiency of channeling was high, with an approximate 50-fold preference for endogenous OMP. By comparison, the degree of channeling for the yeast enzymes, which are soluble and unassociated, was less than 2-fold. The OPRTase-ODCase enzyme complex was solubilized using Triton X-100 in the presence of dimethyl sulfoxide, glycerol, and phosphoribosyldiphosphate. The percentage recovery of the overall enzyme activity was approximately 20%. The degree of channeling was reduced by approximately 10-fold for the solubilized complex. The Km for OMP changed from 7.5 (+/- 1.8) to 1.6 (+/- 0.3) microM in the ODCase reaction. There was no alteration in the Km for orotate in the OPRTase reaction.     

Enzymes of de novo Pyrimidine Biosynthesis in Babesia rodhaini1

ABSTRACT. The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2): dihydroorotase (DHOase: E.C. 3.5.2.3): dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5′-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHasc. OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent K, of 7μM. Dihydro-5-azaorotate inhibited the DHO-DHase (K, 16 μM) and 5-azaorotate (K_i, 21 μM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (K_i, 0.3 μM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product. UMP (K_i, 120 μM) and the purine nucleotide, XMP (K₁, 95 μM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

AS Triazine Acyclonucleosides: Potential Inhibitors of Pyrimidine Enzymes1

Abstract

Seven as-triazine-3,5-dione acyclonucleosides were synthesized and evaluated as inhibitors of orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10), orotidine 5′-monophosphate decarboxylase (ODCase, EC 4.1.2.23), uridine phosphorylase (UrdPase, EC 2.4.2.3), and thymidine phosphorylase (dThdPase, EC 2.4.2.4).     

Crystallization of yeast orotidine 5'-monophosphate decarboxylase complexed with 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 microM dimer) plus 450 microM BMP by microdialysis at 4 degrees C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 microM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 A, c = 385 A. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase: BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 A3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 A resolution over a period of several weeks at room temperature and are isomorphous.     

Enhancing the production of uridine 5′-monophosphate by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using a whole cell biocatalytic process

Attempts were made with success to produce uridine 5′-monophosphate (UMP) from orotic acid by a recombinant Saccharomyces cerevisiae strain pYX212-URA5/BJX12, using the whole cell biocatalytic process. URA5 and URA3 genes, encoding orotate phosphoribosytransferase (OPRTase) and orotidine monophosphate decarboxylase (ODCase), respectively were successfully overexpressed in the industrial yeast strain. As a result, S .cerevisae pYX212-URA5/BJX12 exhibited the highest biocatalytic ability, in contrast with the original industrial yeast strain and S. cerevisae pYX212/BJX12 that overexpressed ODCase only. It indicated that the first step of UMP production from orotic acid is a rate-limiting step. Effects of cultivation for the recombinant strain and biocatalytic reaction conditions on UMP production were also investigated. Cultivating the cells in malt extract medium for 36 h in the exponential phase of growth is in favor of converting orotic acid to UMP. To acquire a higher UMP yield, the conditions of the whole cell biocatalytic reaction were optimized and up to 3.8 g l⁻¹ UMP was produced in 24 h consequently. The yield was fivefold higher than the original UMP yield from the industrial yeast. In addition, the accumulation of 2.6 g l⁻¹ UDP (uridne 5′-diphosphate) in the process demonstrated the possibility for further genetic manipulation: deleting the UMPK (Uridylate Kinase, catalyzing UMP–UDP).     

The mechanism of orotidine 5'-monophosphate decarboxylase: catalysis by destabilization of the substrate

The mechanism of orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) was studied using the decarboxylation of orotic acid analogues as a model system. The rate of decarboxylation of 1,3-dimethylorotic acid and its analogues as well as the stability of their corresponding carbanion intermediates was determined. The results have shown that the stability of the carbanion intermediate is not a critical factor in the rate of decarboxylation. On the other hand, the reaction rate is largely dependent on the equilibrium constant for the formation of a zwitterion. Based on these results, we have proposed a new mechanism in which ODCase catalyzes the decarboxylation of OMP by binding the substrate in a zwitterionic form and providing a destabilizing environment for the carboxylate group of OMP.     

Mapping the active site-ligand interactions of orotidine 5'-monophosphate decarboxylase by crystallography

The crystal structures of orotidine 5'-monophosphate decarboxylases from four different sources have been published recently. However, the detailed mechanism of catalysis of the most proficient enzyme known to date remains elusive. As the ligand-protein interactions at the orotate binding site are crucial to the understanding of this enzyme, we mutated several of the residues surrounding the aromatic part of the substrate, individually and in combination. The ensuing effects on enzyme structure and stability were characterized by X-ray crystallography of inhibitor, product, or substrate complexes and by chemical denaturation with guanidine hydrochloride, respectively. The results are consistent with the residues K42D70K72D75B being charged and forming an 'alternate charge network' around the reactive part of the substrate. In addition to exerting charge-charge repulsion on the orotate carboxylate, Asp70 also makes a crucial contribution to enzyme stability. Consequently, orotidine 5'-monophosphate decarboxylases seem to require the presence of a negative charge at this position for catalysis as well as for correct and stable folding.     

Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster

Summary Eight mutations of the rudimentary-like (r-l) locus were isolated following mutagenesis with ethylmethanesulfonate and inter se crosses revealed three basic complementation groups, using the wing phenotype as an index of complementation. One group consists of three entirely noncomplementing mutants that each specify severe reductions in levels of both r-l-encoded enzymes, orotate phosphoribosyltransferase (OPR-Tase) and orotidylate decarboxylase (ODCase). The other two groups consist of complementing mutants, such that any member of one group fully complements all members of the other group. One of these groups consists of two mutants that each specify severly reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and OD-Case reductions in homoallelic flies, but that appear to contribute OPRTase in certain heteroallelic genotypes. It is concluded that the reciprocal and complementing enzymatic phenotypes of mutants in these two groups account for most instances of genetic trans complementation among r-l mutants. These findings are discussed relative to extant information on OPRTase and OD-Case in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.