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Literature summary extracted from

  • Deay, D.I.; Colvert, K.; Gao, F.; Seibold, S.; Goyal, P.; Aillon, D.; Petillo, P.; Richter, M.
    An active site mutation in 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans changes the substrate specificity in favor of (S)-nicotine (2020), Arch. Biochem. Biophys., 692, 108520 .
    View publication on PubMed

Protein Variants

EC Number Protein Variants Comment Organism
1.5.3.5 D166Q mutation slightly reduces the KM for nicotine, it also reduced enzyme turnover by 5fold with nicotine Paenarthrobacter nicotinovorans
1.5.3.5 additional information adding a maltose-binding protein tag onto the N-terminus markedly increases the thermal stability of the enzyme and increases the observed Vmax value for 6-OH-nicotine by about 4.5fold, due to an increase in the occupancy of the flavin cofactor following expression and purification Paenarthrobacter nicotinovorans
1.5.3.5 R274A/Y311W/C417W combination of mutations predicted to enhance enzyme stability, and mutation Y311W. The triple mutant displays an increased kcat value for nicotine resulting in a comparatively robust oxidation of (S)-nicotine, at the same time reducing the specificity for (S)-OH-nicotine by more than 100fold and increasing that for (S)-nicotine by more than fold Paenarthrobacter nicotinovorans
1.5.3.5 Y311W active site residue Tyr311 forms a hydrogen bond with the hydroxyl group of (S)-6-OH-nicotine within the catalytic pocket. Replacement by a tryptophan residue reduces the kcat for (S)-6-OH-nicotine by more than 6fold Paenarthrobacter nicotinovorans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.3.5 0.06
-
 (S)-6-hydroxynicotine wild-type, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 2.7
-
 (S)-6-hydroxynicotine mutant R274A/Y311W/C417W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 3.4
-
 (S)-6-hydroxynicotine mutant Y311W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 13
-
 (S)-nicotine wild-type, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 13
-
 (S)-nicotine mutant R274A/Y311W/C417W, pH 7.4, 37°C Paenarthrobacter nicotinovorans
1.5.3.5 16
-
 (S)-nicotine mutant R274A/Y311W/C417W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 19
-
 (S)-nicotine mutant Y311W, pH 7.4, 22°C Paenarthrobacter nicotinovorans

Organism

EC Number Organism UniProt Comment Textmining
1.5.3.5 Paenarthrobacter nicotinovorans Q93NH4
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.3.5 (S)-6-hydroxynicotine + H2O + O2
-
 Paenarthrobacter nicotinovorans 1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
-
 ?
1.5.3.5 (S)-nicotine + H2O + O2
-
 Paenarthrobacter nicotinovorans N-methylmyosmine + H2O2
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.3.5 0.1
-
 (S)-nicotine wild-type, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 1.14
-
 (S)-nicotine mutant R274A/Y311W/C417W, pH 7.4, 37°C Paenarthrobacter nicotinovorans
1.5.3.5 1.44
-
 (S)-nicotine mutant Y311W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 2.5
-
 (S)-6-hydroxynicotine mutant R274A/Y311W/C417W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 2.65
-
 (S)-nicotine mutant R274A/Y311W/C417W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 9
-
 (S)-6-hydroxynicotine mutant Y311W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 56
-
 (S)-6-hydroxynicotine wild-type, pH 7.4, 22°C Paenarthrobacter nicotinovorans

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.3.5 FAD the FAD content of freshly prepared wild type enzyme averages 0.17 mol FAD per mol enzyme. The FAD content of MBP-tagged enzyme averages 0.8 mol FAD per mol of enzyme Paenarthrobacter nicotinovorans

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5.3.5 0.0076
-
 (S)-nicotine mutant Y311W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 0.0077
-
 (S)-nicotine wild-type, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 0.0204
-
 (S)-nicotine mutant R274A/Y311W/C417W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 0.0877
-
 (S)-nicotine mutant R274A/Y311W/C417W, pH 7.4, 37°C Paenarthrobacter nicotinovorans
1.5.3.5 9.26
-
 (S)-6-hydroxynicotine mutant R274A/Y311W/C417W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 26.5
-
 (S)-6-hydroxynicotine mutant Y311W, pH 7.4, 22°C Paenarthrobacter nicotinovorans
1.5.3.5 1020
-
 (S)-6-hydroxynicotine wild-type, pH 7.4, 22°C Paenarthrobacter nicotinovorans