Uracil → Orotidine 5′-monophosphate

HRID 1672116Notes

Reaction equation

HRID 1672116 reaction equation
F[C:2]1[C:10](=[O:11])[NH:9][C:8](=[O:12])[NH:7][C:3]=1[C:4]([OH:6])=[O:5].[Cl-].[K+].[OH:15][P:16]([O-:19])([OH:18])=[O:17].[K+].O=C[C@@H:23]([C@H:25]([C@@H:27]([C@@H:29]([CH2:31][OH:32])[OH:30])[OH:28])O)O.N1C=CC(=O)NC1=O.[C@@H]1(N2C=CC(=O)NC2=O)O[C@H](CO)[C@@H](O)[C@H]1O>C(O)C(N)(CO)CO.Cl>[P:16]([O:19][CH2:23][C@H:25]1[O:32][C@@H:31]([N:7]2[C:3]([C:4]([OH:6])=[O:5])=[CH:2][C:10](=[O:11])[NH:9][C:8]2=[O:12])[C@H:29]([OH:30])[C@@H:27]1[OH:28])([OH:18])([OH:15])=[O:17]

Participants

8

Reactant 6

HCID 1174Uracil3×HCID 4873Potassium ChlorideHCID 6029UridineHCID 697115-Fluoroorotic acid2×HCID 107526(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanalHCID 516951Monobasic potassium phosphate

Product 1

HCID 160617Orotidine 5′-monophosphate

Reagents, catalysts & solvents 1

HCID 93573Tromethamine hydrochlorideSolvent

Procedure

After UV irradiation of the suspension of Penicillium multicolor IAM7153 conidia, the spores were coated on a 5-fluoroorotic acid (5-FOA) medium (Boeke et al., Mol. Gen. Genet. 197: 345-346, 1984) and incubated at 27° C. for 6 to 8 days. The 5-FOA-resistant strain grown was picked up respectively onto a minimal medium (0.3% NaNO3: 0.05% KCl, 0.05% MgSO4.7H2O, 0.001% FeSO4.7H2O, 0.1% KH2PO4, 2% glucose, 1.5% agar, pH 6.0) and a minimal medium containing 5 mM uracil respectively, and the media were incubated at 27° C. for 3 to 5 days. The uracil-demanding strain growing only on the minimal medium containing 5 mM uracil was inoculated into a YPM medium containing 5 mM uridine (1% yeast extract, 2% peptone, 2% maltose, pH 6.0) and incubated at 27° C. and 140 rpm for 3 days by shaking culture. 1 g of the hypha and 1 g of sea sand were placed in a mortar cooled on ice and were pulverized sufficiently therein, and 10 ml of 50 mM Tris-HCl (pH 8.0) was added to the mortar, to give a suspension of the hypha. The suspension was centrifuged at 36,000×g for 10 minutes, for separation of the supernatant. The orotic acid phosphoribosyltransferase (OPRTase) activity of the supernatant was determined according to the method of Belser et al., (Meth. Enzymol. 51: 155-167, 1978), and the active strain was regarded as an orotidine-5′-phosphate decarboxylase-deficient strain (IAM7153ΔpyrG) (Horiuchi et al., Curr. Genet. 27: 472-478, 1995).

Workup

  1. customAfter UV irradiation of the suspension of Penicillium multicolor IAM7153 conidia
  2. waitthe media were incubated at 27° C. for 3 to 5 days
  3. customincubated at 27° C.
  4. temperaturecooled on ice
  5. customto give
  6. additiona suspension of the hypha
  7. customg for 10 minutes, for separation of the supernatant

Sources & evidence

ORD record
ord-0026d7eb382d451e8099973e525b7a70
Source dataset
uspto-grants-2011_08
Patent number
US07998721B2

Reaction notes

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