Identification and characterization of human uracil phosphoribosyltransferase (UPRTase)

Li, Jixi; Huang, Shengdong; Chen, Jinzhong; Yang, Zhenxing; Fei, Xiangwei; Zheng, Mou‐Xiong; Ji, Chaoneng; Xie, Yun; Mao, Yumin

doi:10.1007/s10038-007-0129-2

Cited by 27 publications

(36 citation statements)

References 36 publications

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“…Unlike enzymes from the de novo synthesis of UMP, UPRTs have mostly been characterized in lower organisms. Human UPRT has been isolated from the human fetal brain cDNA library [18]. However cloning, expression, and purification yielded a recombinant protein with no detectable UPRT catalytic activity [18].…”

Section: Introductionmentioning

confidence: 99%

“…Human UPRT has been isolated from the human fetal brain cDNA library [18]. However cloning, expression, and purification yielded a recombinant protein with no detectable UPRT catalytic activity [18]. Although there appears to be no solid experimental evidence for the presence of UPRT in humans, UMP can be obtained through uridine phosphorylase [19] reaction followed by uridine kinase activity, and uridine-cytidine kinase enzyme activity [20].…”

Section: Introductionmentioning

confidence: 99%

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Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

et al. 2013

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Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

et al. 2013

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“…DHODH is a key player in the pyrimidine de novo biosynthesis pathway and converts dihydroorotate (DHO) into orotate (24). Both the pyrimidine de novo biosynthesis pathway and the uracil salvage pathway channel into the production of uridine monophosphate (UMP) (25), which is the precursor for all pyrimidine nucleotides needed for RNA (UTP, CTP) and DNA (dTTP, dCTP) synthesis. Leflunomide (Arava) is a Food and Drug Administration (FDA)-approved therapy for rheumatoid arthritis, and its immunosuppressive properties are related to inhibition of T-cell proliferation, which is heavily reliant on pyrimidine pools (26).…”

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confidence: 99%

Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis

Hoffmann

Kunz

Simon

et al. 2011

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

222

207

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Compound A3 was identified in a high-throughput screen for inhibitors of influenza virus replication. It displays broad-spectrum antiviral activity, and at noncytotoxic concentrations it is shown to inhibit the replication of negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus), and retroviruses (HIV). In contrast to mammalian cells, inhibition of viral replication by A3 is absent in chicken cells, which suggests species-specific activity of A3. Correspondingly, the antiviral activity of A3 can be linked to a cellular protein, dihydroorotate dehydrogenase (DHODH), which is an enzyme in the de novo pyrimidine biosynthesis pathway. Viral replication of both RNA and DNA viruses can be restored in the presence of excess uracil, which promotes pyrimidine salvage, or excess orotic acid, which is the product of DHODH in the de novo pyrimidine biosynthesis pathway. Based on these findings, it is proposed that A3 acts by depleting pyrimidine pools, which are crucial for efficient virus replication.

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“…Proteins containing a Uprt domain have been reported in higher multicellular eukaryotes, including humans. However, modifications in the domain associated with uracil binding suggest that higher eukaryote orthologs diverge in function from T. gondii Uprt [43]. Indeed, assays in human and mouse cells have shown that Uprt orthologs are unable to convert uracil to UMP [22].…”

Section: Biosynthetic Labeling Of Newly Transcribed Rnamentioning

confidence: 99%

Temporally and Spatially Restricted Gene Expression Profiling

Tallafuß

Washbourne

Postlethwait

2014

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Identifying gene function in specific cells is critical for understanding the processes that make cells unique. Several different methods are available to isolate actively transcribed RNA or actively translated RNA in specific cells at a chosen time point. Cell-specific mRNA isolation can be accomplished by the expression of transgenes in cells of interest, either directly from a specific promoter or using a modular system such as Gal4/UAS or Cre/lox. All of the methods described in this review, namely thiol-labeling of RNA (TU-tagging or RABT), TRAP (translating ribosome affinity purification) and INTACT (isolation of nuclei tagged in specific cell types), allow next generation sequencing, permitting the identification of enriched gene transcripts within the specific cell-type. We describe here the general concept of each method, include examples, evaluate possible problems related to each technique, and suggest the types of questions for which each method is best suited.

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Identification and characterization of human uracil phosphoribosyltransferase (UPRTase)

Cited by 27 publications

References 36 publications

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis

Temporally and Spatially Restricted Gene Expression Profiling

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