EC Number | Cloned (Comment) | Organism |
---|---|---|
2.4.2.3 | gene up1, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis and tree, quantitative real-time RT-PCR expression analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21trxB(DE3) | Phytophthora capsici |
2.4.2.3 | gene up2, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis and tree, quantitative real-time RT-PCR expression analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21trxB(DE3) | Phytophthora capsici |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
2.4.2.3 | purified recombinant PcUP1 ligand-free and in complex with uracil/ribose-1-phosphate, 2'-deoxyuridine/phosphate or thymidine/phosphate, sitting drop vapor diffusion method, mixing of 0.001 ml of SeMet-PcUP1 as well as native PcUP1 protein at 10 mg/ml with 0.001 ml of reservoir ssolution containing 0.1 M HEPES sodium, pH 7.5, 0.8 M NaH2PO4, and 0.8 M KH2PO4, and equilibration against 0.150 ml of reservoir solution at 10°C. Crystallization of SeMet-PcUP1 and PcUP1 is completed within 5 and 3 days respectively. The PcUP1 crystals in complex with ligands are obtained by soaking crystals with mother liquor containing 10 mM each compound for 24 h, X-ray diffraction structure determination and analysis at 1.57-2.0 A resolution, molecular replacement | Phytophthora capsici |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.4.2.3 | E237K | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | E248K | site-directed mutagenesis, almost inactive mutant enzyme | Phytophthora capsici |
2.4.2.3 | F202A | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | F211A | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | H19D | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | H32D | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | Q206L | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | Q215L | site-directed mutagenesis, the mutant enzyme shows highly reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R104E | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R137E | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R208D | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R217D | site-directed mutagenesis, inactive mutant | Phytophthora capsici |
2.4.2.3 | R264E | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R273E | site-directed mutagenesis, the mutant enzyme shows highly reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R39E | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R59E | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R63E | site-directed mutagenesis, the mutant enzyme shows highly reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | R93E | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | T107A | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
2.4.2.3 | T140A | site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Phytophthora capsici |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.4.2.3 | additional information | - |
additional information | kinetic analysis of UP1 is not consistent with a strong effect of substrate cooperativity | Phytophthora capsici | |
2.4.2.3 | additional information | - |
additional information | kinetic analysis of UP2 is not consistent with a strong effect of substrate cooperativity | Phytophthora capsici | |
2.4.2.3 | 6.96 | - |
uridine | pH and temperature not specified in the publication | Phytophthora capsici | |
2.4.2.3 | 7.55 | - |
uridine | pH and temperature not specified in the publication | Phytophthora capsici |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
2.4.2.3 | additional information | enzyme UP1 has a potential metal binding site but contains no metal ions | Phytophthora capsici |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.2.3 | uridine + phosphate | Phytophthora capsici | - |
uracil + alpha-D-ribose 1-phosphate | - |
r | |
2.4.2.3 | uridine + phosphate | Phytophthora capsici LT1534 | - |
uracil + alpha-D-ribose 1-phosphate | - |
r |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.4.2.3 | no activity in Albugo spp. | - |
- |
- |
2.4.2.3 | no activity in Aphanomyces spp. | - |
- |
- |
2.4.2.3 | no activity in Bremia lactuca | - |
- |
- |
2.4.2.3 | no activity in Hyaloperonospora arabidopsidis | - |
- |
- |
2.4.2.3 | no activity in Saprolegnia spp. | - |
- |
- |
2.4.2.3 | Phytophthora capsici | A0A410UCT3 | - |
- |
2.4.2.3 | Phytophthora capsici | A0A6G6VYG7 | - |
- |
2.4.2.3 | Phytophthora capsici LT1534 | A0A410UCT3 | - |
- |
2.4.2.3 | Phytophthora capsici LT1534 | A0A6G6VYG7 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.4.2.3 | recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21trxB(DE3) by nickel affinity chromatography and gel filtration | Phytophthora capsici |
EC Number | Reaction | Comment | Organism | Reaction ID |
---|---|---|---|---|
2.4.2.3 | uridine + phosphate = uracil + alpha-D-ribose 1-phosphate | mutational analysis supports for the push-pull model of catalysis, proposed catalytic mechanism of PcUP1, modeling, overview. Deprotonation of phosphate by Arg104, increases the negative charge on the phosphate oxygen, leading to repulsion between the electrons of phosphate oxygen and electron pairs of the endocylic ribosyl oxygen and the formation of an intermediate oxocarbenium ion. At the same time, the Gln206, Arg208 and Arg264-N1 hydrogen network-bond pulls electrons from the glycosidic bond onto the pyrimidine ring. These two actions work in concert to weaken the glycosidic bond. Arg39 pivots, to physically push the phosphate closer to the ribose moiety. After glycosidic bond cleavage, ribose-1-phosphate dissociates from the active pocket, and then an active water reprotonates the negatively charged purine. The final step is to release the neutral purine base | Phytophthora capsici |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.2.3 | uridine + phosphate | - |
Phytophthora capsici | uracil + alpha-D-ribose 1-phosphate | - |
r | |
2.4.2.3 | uridine + phosphate | - |
Phytophthora capsici LT1534 | uracil + alpha-D-ribose 1-phosphate | - |
r |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
2.4.2.3 | homodimer | although the homodimer's conformation of PcUP1 is equivalent to the dimer unit in the typical NP-I subfamily, it is not possible to assemble three PcUP1 dimers into the canonical hexamer as a result of a 16-amino-acid insertion in the sequence of PcUP1. This Phytophthora capsici-specific insert creates an additional secondary structural element, that protrudes into the space that would be occupied by the neighboring dimer of the canonical NP-1 hexamer, thus sterically blocking trimerization of the dimers. Trypanosoma brucei TbUP and human HsUPP1 also harbor hexamer-blocking insertions. The strictly ear-shaped conserved catalytic pocket with positive charge of PcUP1 is located on the monomer-monomer hydrophobic interface | Phytophthora capsici |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.4.2.3 | PcUP1 | - |
Phytophthora capsici |
2.4.2.3 | PcUP2 | - |
Phytophthora capsici |
2.4.2.3 | UP1 | - |
Phytophthora capsici |
2.4.2.3 | UP2 | - |
Phytophthora capsici |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
2.4.2.3 | 25 | - |
assay at | Phytophthora capsici |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.4.2.3 | 982 | - |
uridine | pH and temperature not specified in the publication | Phytophthora capsici | |
2.4.2.3 | 1188 | - |
uridine | pH and temperature not specified in the publication | Phytophthora capsici |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
2.4.2.3 | 7.5 | - |
assay at | Phytophthora capsici |
EC Number | Organism | Comment | Expression |
---|---|---|---|
2.4.2.3 | Phytophthora capsici | PcUP1 is upregulated 300fold within 90 min after infection of pepper leaves | up |
2.4.2.3 | Phytophthora capsici | PcUP2 is upregulated 500fold within 90 min after infection of pepper leaves | up |
EC Number | General Information | Comment | Organism |
---|---|---|---|
2.4.2.3 | evolution | phylogenetic analysis of UPs in oomycetes | Phytophthora capsici |
2.4.2.3 | metabolism | uridine phosphorylase (UP) is a key enzyme of pyrimidine salvage pathways that enables the recycling of endogenous or exogenous-supplied pyrimidines and plays an important intracellular metabolic role | Phytophthora capsici |
2.4.2.3 | additional information | strict conservation of UP1 key residues in the binding pocket, structure analysis of PcUP1 with bound ligands, active site structure and substrate binding, overview | Phytophthora capsici |
EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.4.2.3 | 141.1 | - |
uridine | pH and temperature not specified in the publication | Phytophthora capsici | |
2.4.2.3 | 157.4 | - |
uridine | pH and temperature not specified in the publication | Phytophthora capsici |