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Literature summary extracted from

  • Theriot, C.M.; Du, X.; Tove, S.R.; Grunden, A.M.
    Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures (2010), Appl. Microbiol. Biotechnol., 87, 1715-1726.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.4.13.9 DNA and amino acid sequence determination and analysis, expression of wild-type and mutant enzymes in Escherichia coli strain JD1 (lambdaDE3) Pyrococcus furiosus

Protein Variants

EC Number Protein Variants Comment Organism
3.4.13.9 G39E site-directed mutagenesis, the mutant shows altered kinetics and temperature profile Pyrococcus furiosus
3.4.13.9 G39E at 35°C, 70°C and 100°C the mutant exhibits higher Vmax and kcat values than wild-type prolidase for Met-Pro. kcat/Km for Met-Pro is 70% of wild-type value at 35°C, kcat/Km for Met-Pro is 80% of wild-type value at 70°C, kcat/Km for Met-Pro is 1.2fold higher than wild-type value at 100°C. Relative specific activity towards Met-Pro at 100°C is 103% of wild-type activity. Relative specific activity towards Leu-Pro at 100°C is 85% of wild-type activity. Relative specific activity towards Phe-Pro at 100°C is 60% of wild-type activity. Relative specific activity towards Ala-Pro at 100°C is 35% of wild-type activity. Relative specific activity towards Gly-Pro at 100°C is 37% of wild-type activity. Relative specific activity towards Arg-Pro at 100°C is 132% of wild-type activity. Catalytic activity of the mutant enzyme has similar response to changes in pH as wild-type enzyme and shows optimal activity at pH 6.0, although the activity is 60% of wild-type activity Pyrococcus furiosus
3.4.13.9 additional information the use of Pyrococcus furiosus prolidase for organophosphorus nerve agent decontamination is restricted by the fact that this enzyme displays a narrow functional temperature range. Like many other enzymes isolated from hyperthermophiles, Pyrococcus furiosus prolidase has only 50% activity at 80°C and displays little activity at temperatures below 50°C. Therefore a random-mutated Pyrococcus furiosus prolidase gene library is constructed and screened for production of mutants with increased activity at room temperature while maintaining thermostability. The mutant enzyme shows increased activity over the pH range of 5–7. At pH 5.0, the mutant activity is 1.6fold higher than wild-type activity, and at pH 7.0, it is 1.2fold higher than wild type Pyrococcus furiosus
3.4.13.9 R19G/G39E/K71E/S229T site-directed mutagenesis, the mutant shows altered kinetics and temperature profile Pyrococcus furiosus
3.4.13.9 R19G/G39E/K71E/S229T mutant enzyme shows a higher activity than wild-type enzyme over a broad range of temperatures, the thermostability of the mutant enzymes is less compared to wild type. At 35°C, 70°C and 100°C the mutant exhibits higher Vmax and kcat values than wild-type prolidase for Met-Pro. kcat/Km for Met-Pro is 1.2fold higher than wild-type value at 35°C, kcat/Km for Met -Pro is 1.8fold higher than wild-type value at 70°C, kcat/Km for Met-Pro is 2.5fold higher than wild-type value at 100°C. Relative specific activity towards Met-Pro at 100°C is 143% of wild-type activity. Relative specific activity towards Leu-Pro at 100°C is 79% of wild-type activity. Relative specific activity towards Phe-Pro at 100°C is 122% of wild-type activity. Relative specific activity towards Ala-Pro at 100°C is 38% of wild-type activity. Relative specific activity towards Gly-Pro at 100°C is 10% of wild-type activity. Relative specific activity towards Arg-Pro at 100°C is 112% of wild-type activity Pyrococcus furiosus
3.4.13.9 R19G/K71E/S229 site-directed mutagenesis, the mutant shows altered kinetics and temperature profile Pyrococcus furiosus
3.4.13.9 R19G/K71E/S229T mutant enzyme shows a higher activity than wild-type enzyme over a broad range of temperatures, the thermostability of the mutant enzymes is less compared to wild type. At 35°C, 70°C and 100°C the mutant exhibits higher Vmax and kcat values than wild-type prolidase for Met-Pro. kcat/Km for Met-Pro is 1.1fold higher than wild-type value at 35°C, kcat/Km for Met -Pro comparable to wild-type value at 70°C, kcat/Km for Met-Pro is 2.4fold higher than wild-type value at 100°C. Relative specific activity towards Met-Pro at 100°C is 137% of wild-type activity. Relative specific activity towards Leu-Pro at 100°C is 169% of wild-type activity. Relative specific activity towards Phe-Pro at 100°C is 97% of wild-type activity. Relative specific activity towards Ala-Pro at 100°C is 95% of wild-type activity. Relative specific activity towards Gly-Pro at 100°C is 47% of wild-type activity. Relative specific activity towards Arg-Pro at 100°C is 101% of wild-type activity. Catalytic activity of the mutant enzyme has similar response to changes in pH as wild-type enzyme and shows optimal activity at pH 7.0, although the activity is 89% of wild-type activity Pyrococcus furiosus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.4.13.9 5.7
-
Met-Pro pH 7.0, 70°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 5.7
-
Met-Pro pH 7.0, 70°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 6.4
-
Met-Pro pH 7.0, 35°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 6.4
-
Met-Pro pH 7.0, 35°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 6.8
-
Met-Pro pH 7.0, 70°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 6.8
-
Met-Pro pH 7.0, 70°C, wild-type enzyme G39E Pyrococcus furiosus
3.4.13.9 7.5
-
Met-Pro pH 7.0, 70°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 7.5
-
Met-Pro pH 7.0, 70°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 7.9
-
Met-Pro pH 7.0, 35°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 7.9
-
Met-Pro pH 7.0, 35°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 7.95
-
Met-Pro pH 7.0, 100°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 7.95
-
Met-Pro pH 7.0, 100°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 9.2
-
Met-Pro pH 7.0, 70°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 9.2
-
Met-Pro pH 7.0, 70°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 11.1
-
Met-Pro pH 7.0, 100°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 11.1
-
Met-Pro pH 7.0, 100°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 11.3
-
Met-Pro pH 7.0, 35°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 11.3
-
Met-Pro pH 7.0, 35°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 13
-
Met-Pro pH 7.0, 35°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 13
-
Met-Pro pH 7.0, 35°C, mutant enzyme G39E Pyrococcus furiosus
3.4.13.9 13.7
-
Met-Pro pH 7.0, 100°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 13.7
-
Met-Pro pH 7.0, 100°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 14.5
-
Met-Pro pH 7.0, 100°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 14.5
-
Met-Pro pH 7.0, 100°C, wild-type enzyme G39E Pyrococcus furiosus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.4.13.9 Co2+ activates Pyrococcus furiosus

Organism

EC Number Organism UniProt Comment Textmining
3.4.13.9 Pyrococcus furiosus
-
-
-
3.4.13.9 Pyrococcus furiosus P81535
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.4.13.9 recombinant wild-type and mutant enzymes from Escherichia coli strain JD1 (lambdaDE3) by heat treatment at 80°C for 30 min, hydrophobic interaction chromatography, and anion exchange chromatography Pyrococcus furiosus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.4.13.9 4-nitrophenyl soman + H2O
-
Pyrococcus furiosus ?
-
?
3.4.13.9 Ala-Pro + H2O
-
Pyrococcus furiosus Ala + Pro
-
?
3.4.13.9 Arg-Pro + H2O
-
Pyrococcus furiosus Arg + Pro
-
?
3.4.13.9 Gly-Pro + H2O
-
Pyrococcus furiosus Gly + Pro
-
?
3.4.13.9 Leu-Pro + H2O
-
Pyrococcus furiosus Leu + Pro
-
?
3.4.13.9 Met-Pro + H2O
-
Pyrococcus furiosus Met + Pro
-
?
3.4.13.9 Met-Pro + H2O preferred substrate of wild-type and mutant enzymes Pyrococcus furiosus Met + Pro
-
?
3.4.13.9 Phe-Pro + H2O
-
Pyrococcus furiosus Phe + Pro
-
?
3.4.13.9 Pro-Ala + H2O
-
Pyrococcus furiosus Pro + Ala
-
?

Synonyms

EC Number Synonyms Comment Organism
3.4.13.9 prolidase
-
Pyrococcus furiosus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.4.13.9 10 100 activities of wild-type and mutant enzymes at 100°C Pyrococcus furiosus

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3.4.13.9 35 100 highest activities of wild-type and mutant enzymes at 100°C Pyrococcus furiosus

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.4.13.9 90
-
mutants K71E/S229T and R19G/G39E/K71E/S229T loose 50% activity after 4 and 3 h, respectively, whereas mutant G39E does not show this loss of activity until 14 h, and the wild-type enzyme retains 50% activity after 21 h Pyrococcus furiosus
3.4.13.9 90
-
R19G/K71E/S229T loses 50% of its activity after 4 h, R19G/G39E/K71E/S229T loses 50% of its activity after 3 h, G39E loses 50% of its activity after 14 h, wild-type enzyme loses 50% of its activity after 21 h Pyrococcus furiosus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.4.13.9 123
-
Met-Pro pH 7.0, 35°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 123
-
Met-Pro pH 7.0, 35°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 167
-
Met-Pro pH 7.0, 35°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 167
-
Met-Pro pH 7.0, 35°C, mutant enzyme G39E Pyrococcus furiosus
3.4.13.9 171
-
Met-Pro pH 7.0, 35°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 171
-
Met-Pro pH 7.0, 35°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 173
-
Met-Pro pH 7.0, 35°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 173
-
Met-Pro pH 7.0, 35°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 767
-
Met-Pro pH 7.0, 70°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 767
-
Met-Pro pH 7.0, 70°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 1017
-
Met-Pro pH 7.0, 70°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 1017
-
Met-Pro pH 7.0, 70°C, wild-type enzyme G39E Pyrococcus furiosus
3.4.13.9 1217
-
Met-Pro pH 7.0, 70°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 1217
-
Met-Pro pH 7.0, 70°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 2104
-
Met-Pro pH 7.0, 100°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 2104
-
Met-Pro pH 7.0, 100°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 2416
-
Met-Pro pH 7.0, 70°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 2416
-
Met-Pro pH 7.0, 70°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 4594
-
Met-Pro pH 7.0, 100°C, wild-type enzyme G39E Pyrococcus furiosus
3.4.13.9 6396
-
Met-Pro pH 7.0, 100°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 6396
-
Met-Pro pH 7.0, 100°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 6995
-
Met-Pro pH 7.0, 100°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 7481
-
Met-Pro pH 7.0, 100°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 7481
-
Met-Pro pH 7.0, 100°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.4.13.9 6
-
-
Pyrococcus furiosus
3.4.13.9 7
-
assay at Pyrococcus furiosus

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.4.13.9 4 10
-
Pyrococcus furiosus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.4.13.9 13
-
Met-Pro pH 7.0, 35°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 13
-
Met-Pro pH 7.0, 35°C, mutant enzyme G39E Pyrococcus furiosus
3.4.13.9 15
-
Met-Pro pH 7.0, 35°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 15
-
Met-Pro pH 7.0, 35°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 19
-
Met-Pro pH 7.0, 35°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 19
-
Met-Pro pH 7.0, 35°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 22
-
Met-Pro pH 7.0, 35°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 22
-
Met-Pro pH 7.0, 35°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 132
-
Met-Pro pH 7.0, 70°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 132
-
Met-Pro pH 7.0, 70°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 135
-
Met-Pro pH 7.0, 70°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 135
-
Met-Pro pH 7.0, 70°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 150
-
Met-Pro pH 7.0, 70°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 150
-
Met-Pro pH 7.0, 70°C, wild-type enzyme G39E Pyrococcus furiosus
3.4.13.9 266
-
Met-Pro pH 7.0, 100°C, recombinant wild-type enzyme Pyrococcus furiosus
3.4.13.9 266
-
Met-Pro pH 7.0, 100°C, wild-type enzyme Pyrococcus furiosus
3.4.13.9 317
-
Met-Pro pH 7.0, 100°C, recombinant mutant G39E Pyrococcus furiosus
3.4.13.9 317
-
Met-Pro pH 7.0, 100°C, wild-type enzyme G39E Pyrococcus furiosus
3.4.13.9 322
-
Met-Pro pH 7.0, 70°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 322
-
Met-Pro pH 7.0, 70°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 467
-
Met-Pro pH 7.0, 100°C, recombinant mutant R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 467
-
Met-Pro pH 7.0, 100°C, mutant enzyme R19G/K71E/S229T Pyrococcus furiosus
3.4.13.9 674
-
Met-Pro pH 7.0, 100°C, recombinant mutant R19G/G39E/K71E/S229T Pyrococcus furiosus
3.4.13.9 674
-
Met-Pro pH 7.0, 100°C, mutant enzyme R19G/G39E/K71E/S229T Pyrococcus furiosus