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Literature summary extracted from

  • Mahor, D.; Priyanka, A.; Prasad, G.S.; Thakur, K.G.
    Functional and structural characterization of purine nucleoside phosphorylase from Kluyveromyces lactis and its potential applications in reducing purine content in food (2016), PLoS ONE, 11, e0164279 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.2.1 sequence comparisons, recombinant expression of N- and C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli Kluyveromyces lactis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.2.1 purified enzyme KlacPNP in complex with hypoxanthine, screening of recombinant N- and C-terminally His6-tagged wild-type enzyme, vapor diffusion sitting drop, mixing of 0.00125 ml of 8 mg/ml of protein in 25 mM Tris-HCl, 100 mM NaCl, pH 8.0, with 0.00125 ml of mother liquor consisting of 0.1 M sodium acetate trihydrate, pH 4.6, and 8% PEG 4000, X-ray diffraction structure determination and analysis at 1.97 A resolution, molecular replacement using the human PNP as a search template (PDB ID 3PHB) Kluyveromyces lactis

Protein Variants

EC Number Protein Variants Comment Organism
2.4.2.1 N256D site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine Kluyveromyces lactis
2.4.2.1 N256E site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine Kluyveromyces lactis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.2.1 0.0103
-
 adenosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 0.021
-
 Inosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 0.144
-
 Inosine recombinant mutant N256E, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 0.222
-
 Inosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 0.5
-
 guanosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 0.536
-
 Xanthosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 0.555
-
 Xanthosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 0.842
-
 guanosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.4.2.1 105000
-
 recombinant His-tagged enzyme, gel filtration Kluyveromyces lactis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.2.1 guanosine + phosphate Kluyveromyces lactis
-
 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate Kluyveromyces lactis CBS 2359
-
 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate Kluyveromyces lactis NRRL Y-1140
-
 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate Kluyveromyces lactis DSM 70799
-
 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate Kluyveromyces lactis WM37
-
 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate Kluyveromyces lactis ATCC 8585
-
 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate Kluyveromyces lactis NBRC 1267
-
 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate Kluyveromyces lactis
-
 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate Kluyveromyces lactis CBS 2359
-
 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate Kluyveromyces lactis NRRL Y-1140
-
 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate Kluyveromyces lactis DSM 70799
-
 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate Kluyveromyces lactis WM37
-
 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate Kluyveromyces lactis ATCC 8585
-
 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate Kluyveromyces lactis NBRC 1267
-
 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 purine ribonucleoside + phosphate Kluyveromyces lactis
-
 purine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 xanthosine + phosphate Kluyveromyces lactis
-
 xanthine + alpha-D-ribose 1-phosphate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
2.4.2.1 Kluyveromyces lactis Q6CSZ6
-
-
2.4.2.1 Kluyveromyces lactis ATCC 8585 Q6CSZ6
-
-
2.4.2.1 Kluyveromyces lactis CBS 2359 Q6CSZ6
-
-
2.4.2.1 Kluyveromyces lactis DSM 70799 Q6CSZ6
-
-
2.4.2.1 Kluyveromyces lactis NBRC 1267 Q6CSZ6
-
-
2.4.2.1 Kluyveromyces lactis NRRL Y-1140 Q6CSZ6
-
-
2.4.2.1 Kluyveromyces lactis WM37 Q6CSZ6
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.2.1 recombinant N- and C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, dialysis, ultrafiltration, and gel filtration Kluyveromyces lactis

Storage Stability

EC Number Storage Stability Organism
2.4.2.1 purified recombinant His-tagged enzyme at 25 mg/ml is stable for months when stored at 8°C in an elution buffer containing 25 mM Tris-HCl, 100 mM NaCl, 10% glycerol, pH 7.0, up to 65% activity is retained after storage under these conditions for over 30 days Kluyveromyces lactis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.2.1 adenosine + phosphate
-
 Kluyveromyces lactis adenine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 adenosine + phosphate
-
 Kluyveromyces lactis CBS 2359 adenine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 adenosine + phosphate
-
 Kluyveromyces lactis NRRL Y-1140 adenine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 adenosine + phosphate
-
 Kluyveromyces lactis DSM 70799 adenine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 adenosine + phosphate
-
 Kluyveromyces lactis WM37 adenine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 adenosine + phosphate
-
 Kluyveromyces lactis ATCC 8585 adenine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 adenosine + phosphate
-
 Kluyveromyces lactis NBRC 1267 adenine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate
-
 Kluyveromyces lactis guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate
-
 Kluyveromyces lactis CBS 2359 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate
-
 Kluyveromyces lactis NRRL Y-1140 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate
-
 Kluyveromyces lactis DSM 70799 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate
-
 Kluyveromyces lactis WM37 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate
-
 Kluyveromyces lactis ATCC 8585 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 guanosine + phosphate
-
 Kluyveromyces lactis NBRC 1267 guanine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate
-
 Kluyveromyces lactis hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate preferred substrate Kluyveromyces lactis hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate
-
 Kluyveromyces lactis CBS 2359 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate preferred substrate Kluyveromyces lactis CBS 2359 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate
-
 Kluyveromyces lactis NRRL Y-1140 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate preferred substrate Kluyveromyces lactis NRRL Y-1140 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate
-
 Kluyveromyces lactis DSM 70799 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate preferred substrate Kluyveromyces lactis DSM 70799 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate
-
 Kluyveromyces lactis WM37 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate preferred substrate Kluyveromyces lactis WM37 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate
-
 Kluyveromyces lactis ATCC 8585 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate preferred substrate Kluyveromyces lactis ATCC 8585 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate
-
 Kluyveromyces lactis NBRC 1267 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 inosine + phosphate preferred substrate Kluyveromyces lactis NBRC 1267 hypoxanthine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 additional information no significant activity towards adenosine. Substrate binding structure analysis, active site structure comparisons, overview Kluyveromyces lactis ?
-
-
2.4.2.1 additional information no significant activity towards adenosine. Substrate binding structure analysis, active site structure comparisons, overview Kluyveromyces lactis CBS 2359 ?
-
-
2.4.2.1 additional information no significant activity towards adenosine. Substrate binding structure analysis, active site structure comparisons, overview Kluyveromyces lactis NRRL Y-1140 ?
-
-
2.4.2.1 additional information no significant activity towards adenosine. Substrate binding structure analysis, active site structure comparisons, overview Kluyveromyces lactis DSM 70799 ?
-
-
2.4.2.1 additional information no significant activity towards adenosine. Substrate binding structure analysis, active site structure comparisons, overview Kluyveromyces lactis WM37 ?
-
-
2.4.2.1 additional information no significant activity towards adenosine. Substrate binding structure analysis, active site structure comparisons, overview Kluyveromyces lactis ATCC 8585 ?
-
-
2.4.2.1 additional information no significant activity towards adenosine. Substrate binding structure analysis, active site structure comparisons, overview Kluyveromyces lactis NBRC 1267 ?
-
-
2.4.2.1 purine ribonucleoside + phosphate
-
 Kluyveromyces lactis purine + alpha-D-ribose 1-phosphate
-
 r
2.4.2.1 xanthosine + phosphate
-
 Kluyveromyces lactis xanthine + alpha-D-ribose 1-phosphate
-
 r

Subunits

EC Number Subunits Comment Organism
2.4.2.1 homotrimer
-
 Kluyveromyces lactis

Synonyms

EC Number Synonyms Comment Organism
2.4.2.1 KlacPNP
-
 Kluyveromyces lactis
2.4.2.1 KLLA0_C16621g locus name Kluyveromyces lactis
2.4.2.1 PNP
-
 Kluyveromyces lactis
2.4.2.1 purine nucleoside phosphorylase
-
 Kluyveromyces lactis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.2.1 25
-
 assay at Kluyveromyces lactis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4.2.1 10.4
-
 guanosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 16.8
-
 Xanthosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 22.8
-
 adenosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 23.3
-
 Xanthosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 25.1
-
 Inosine recombinant mutant N256E, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 29.7
-
 guanosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 34.9
-
 Inosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 43.9
-
 Inosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.2.1 7
-
 assay at Kluyveromyces lactis

General Information

EC Number General Information Comment Organism
2.4.2.1 metabolism purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP) is a key enzyme involved in the purine degradation pathway, overview Kluyveromyces lactis
2.4.2.1 additional information KlacPNP and KlacPNPN256D reduce purine content in a beer sample Kluyveromyces lactis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.4.2.1 12.4
-
 guanosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 30.3
-
 Xanthosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 43.5
-
 Xanthosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 59.4
-
 guanosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 157.2
-
 Inosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 174.3
-
 Inosine recombinant mutant N256E, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 2090.5
-
 Inosine recombinant wild-type enzyme, pH 7.0, 25°C Kluyveromyces lactis
2.4.2.1 2213.6
-
 adenosine recombinant mutant N256D, pH 7.0, 25°C Kluyveromyces lactis