Intrinsic Activity and Stability of Bifunctional Human UMP Synthase and Its Two Separate Catalytic Domains, Orotate Phosphoribosyltransferase and Orotidine-5′-phosphate Decarboxylase

Yablonski, Michael J.; Pasek, Daniel A.; Han, Byoung Don; Jones, Mary Ellen; Traut, Thomas W.

doi:10.1074/jbc.271.18.10704

Cited by 46 publications

(66 citation statements)

References 31 publications

(24 reference statements)

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“…Together, these three H-bonds tether the BMP ribosyl moiety to the enzyme, constraining the position of the pyrimidine ring within the active site. The interaction between the ligand's ribosyl OH groups and the side chains of Asp-96* and Thr-100* of the second subunit is consistent with the proposal that the active species of yeast ODCase is a dimer similar to that shown for the bifunctional human uridine 5Ј-phosphate synthase and its ODCase domain (14).…”

Section: Resultssupporting

confidence: 68%

Anatomy of a proficient enzyme: The structure of orotidine 5′-monophosphate decarboxylase in the presence and absence of a potential transition state analog

Miller¹,

Hassell

Wolfenden

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

194

276

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Orotidine 5 -phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme. The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5 -phosphate, at a resolution of 2.1 Å and 2.4 Å, respectively. Orotidine 5 -phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel. The binding of 6-hydroxyuridine 5 -phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product. In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state. Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation.O rotidine 5Ј-phosphate decarboxylase (ODCase) (EC 4.1.1.23) is responsible for de novo synthesis of uridine 5Ј-phosphate, an essential precursor of RNA and DNA. In neutral solution, orotidine 5Ј-monophosphate (OMP) undergoes spontaneous decarboxylation to uridine 5Ј-phosphate with a half-time of 78 million years (1). At the ODCase active site, the same reaction proceeds with a half-time of 18 msec (2). Comparison of k cat ͞K m with k non indicates that ODCase surpasses other enzymes in its proficiency ʈ as a catalyst, achieving a remarkable affinity for the altered substrate in the transition state (1). In addition to surmounting this formidable kinetic barrier, the ODCase reaction is of special interest in view of its lack of precedent in biological chemistry. The substrate is devoid of an effective repository for the negative charge that is generated at C-6 when CO 2 is eliminated, yet the enzyme functions without metals or other cofactors. Enzymatic decarboxylation of OMP is also remarkable in the importance (for catalysis) of a seemingly irrelevant part of the substrate. By its presence, the 5Ј-phosphoryl group contributes a factor of Ϸ10 8 -fold to k cat ͞K m , in spite of its considerable distance from the site of chemical transformation of the substrate (3). As a first step toward understanding these unusual properties, we have investigated the crystal structure of recombinant Saccharomyces cerevisiae ODCase alone and in complex with a postulated transition state analogue, 6-hydroxyuridine 5Ј-phosphate (BMP) (K i ϭ 9 ϫ 10 Ϫ12 M (4)]. Materials and MethodsRecombinant yeast ODCase was expressed in Escherichia coli SS6130 (pBGM88) and was purified as described (5). Crystals of the native enzyme were grown a...

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Section: Resultssupporting

confidence: 68%

Anatomy of a proficient enzyme: The structure of orotidine 5′-monophosphate decarboxylase in the presence and absence of a potential transition state analog

Miller¹,

Hassell

Wolfenden

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

194

276

View full text Add to dashboard Cite

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“…S2 in the supplemental material). The accumulation of orotidine-monophosphate was not monitored; therefore, we hypothesize that compound 1 may inhibit orotate phosphoribosyltransferase (OPRTase) activity, which is executed by a dual functional enzyme, UMP synthetase (Umps) (20). Nevertheless, the addition of orotate lessened the antiviral effect of compound 1 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State

Chung

Golden

Adcock

et al. 2016

Antimicrob Agents Chemother

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Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons.

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“…The activity of UMPS was analyzed by radioactive assay according to the procedure of Yablonski et al (1996). UK and UPRT activities were determined essentially according to the procedure of Katahira and Ashihara (2002), with the following modifications: longer incubation times were used after it had been determined that the reaction was linear for at least 4 h, and the reactions were heat-inactivated (958C for 5 min) rather than perchlorate-inactivated.…”

Section: Analysis Of Enzyme Activitiesmentioning

confidence: 99%

Inhibition of de Novo Pyrimidine Synthesis in Growing Potato Tubers Leads to a Compensatory Stimulation of the Pyrimidine Salvage Pathway and a Subsequent Increase in Biosynthetic Performance

et al. 2005

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Pyrimidine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. In this study, we investigate the influence of a decreased expression of UMP synthase (UMPS), a key enzyme in the pathway of de novo pyrimidine synthesis, on biosynthetic processes in growing potato (Solanum tuberosum) tubers. Transgenic plants were generated expressing UMPS in the antisense orientation under the control of the tuber-specific patatin promoter. Lines were selected with markedly decreased expression of UMPS in the tubers. Decreased expression of UMPS restricted the use of externally supplied orotate for de novo pyrimidine synthesis in tuber tissue, whereas the uridine-salvaging pathway was stimulated. This shift in the pathways of UMP synthesis was accompanied by increased levels of tuber uridine nucleotides, increased fluxes of [ 14 C]sucrose to starch and cell wall synthesis, and increased amounts of starch and cell wall components in the tubers, whereas there were no changes in uridine nucleotide levels in leaves. Decreased expression of UMPS in tubers led to an increase in transcript levels of carbamoylphosphate synthase, uridine kinase, and uracil phosphoribosyltransferase, the latter two encoding enzymes in the pyrimidine salvage pathways. Thus, the results show that antisense inhibition of the de novo pathway of pyrimidine synthesis leads to a compensatory stimulation of the less energyconsuming salvage pathways, probably via increased expression and activity of uridine kinase and uracil phosphoribosyltransferase. This results in increased uridine nucleotide pool levels in tubers and improved biosynthetic performance.

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Intrinsic Activity and Stability of Bifunctional Human UMP Synthase and Its Two Separate Catalytic Domains, Orotate Phosphoribosyltransferase and Orotidine-5′-phosphate Decarboxylase

Cited by 46 publications

References 31 publications

Anatomy of a proficient enzyme: The structure of orotidine 5′-monophosphate decarboxylase in the presence and absence of a potential transition state analog

Anatomy of a proficient enzyme: The structure of orotidine 5′-monophosphate decarboxylase in the presence and absence of a potential transition state analog

Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State

Inhibition of de Novo Pyrimidine Synthesis in Growing Potato Tubers Leads to a Compensatory Stimulation of the Pyrimidine Salvage Pathway and a Subsequent Increase in Biosynthetic Performance

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