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Literature summary extracted from

  • Yang, C.; Li, J.; Huang, Z.; Zhang, X.; Gao, X.; Zhu, C.; Morris, P.F.; Zhang, X.
    Structural and catalytic analysis of two diverse uridine phosphorylases in Phytophthora capsici (2020), Sci. Rep., 10, 9051 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

Crystallization (Commentary)

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

Protein Variants

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

KM Value [mM]

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

Metals/Ions

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

Natural Substrates/ Products (Substrates)

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

Organism

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
-
-

Purification (Commentary)

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

Reaction

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

Substrates and Products (Substrate)

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

Subunits

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

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.2.3 25
-
assay at Phytophthora capsici

Turnover Number [1/s]

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

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.2.3 7.5
-
assay at Phytophthora capsici

Expression

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

General Information

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

kcat/KM [mM/s]

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