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Literature summary extracted from

  • Mensitieri, F.; De Lise, F.; Strazzulli, A.; Moracci, M.; Notomista, E.; Cafaro, V.; Bedini, E.; Sazinsky, M.H.; Trifuoggi, M.; Di Donato, A.; Izzo, V.
    Structural and functional insights into RHA-P, a bacterial GH106 alpha-L-rhamnosidase from Novosphingobium sp. PP1Y (2018), Arch. Biochem. Biophys., 648, 1-11 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.40 the recombinant protein is fused to a C-terminal His-tag and expressed in Escherichia coli BL21(DE3) Novosphingobium sp. PP1Y

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.40 D503A no detectable activity Novosphingobium sp. PP1Y
3.2.1.40 D552A kcat/KM value is 65% of the wild-type value. This reduced catalytic efficiency is mainly due to a significative variation in the turnover number, defined by the kcat value, for which a 34% decrease is observed Novosphingobium sp. PP1Y
3.2.1.40 D552A mutant enzyme shows 47.9% of residual activity Novosphingobium sp. PP1Y
3.2.1.40 D763A kcat/KM value is 21% of the wild-type value. A 82% decrease in kcat is retrieved for the mutant enzyme, while the KM is similar within the experimental error Novosphingobium sp. PP1Y
3.2.1.40 D763A mutant enzyme shows 15.8% of residual activity Novosphingobium sp. PP1Y
3.2.1.40 E506A mutant enzyme shows 0.05% of residual activity Novosphingobium sp. PP1Y
3.2.1.40 E644A no detectable activity Novosphingobium sp. PP1Y

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.40 0.031
-
rutin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 0.067
-
hesperidin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 0.138
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, mutant enzyme D763A Novosphingobium sp. PP1Y
3.2.1.40 0.15
-
quercitrin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 0.157
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, wild-type enzyme Novosphingobium sp. PP1Y
3.2.1.40 0.159
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, mutant enzyme D552A Novosphingobium sp. PP1Y
3.2.1.40 0.59
-
naringin pH 6.9, 37°C Novosphingobium sp. PP1Y

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.2.1.40 Ca2+ EDTA-treated enzyme recovers its activity in the presence of divalent cations such as Mg2+ and Ca2+, but with an apparent higher efficiency when calcium is used. Almost total recovery of activity in the presence of a 3000-fold excess of Ca2+ compared to the 56% obtained with a similar excess of Mg2+. Five residues contribute to the binding of the Ca2+ ion: E593, E538, D458, S459 and E561 Novosphingobium sp. PP1Y
3.2.1.40 Mg2+ EDTA-treated enzyme recovers its activity in the presence of divalent cations such as Mg2+ and Ca2+, but with an apparent higher efficiency when calcium is used. Almost total recovery of activity in the presence of a 3000-fold excess of Ca2+ compared to the 56% obtained with a similar excess of Mg2+ Novosphingobium sp. PP1Y

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.2.1.40 12000
-
-
Novosphingobium sp. PP1Y

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.40 Novosphingobium sp. PP1Y F6IEX3
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.40
-
Novosphingobium sp. PP1Y

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.40 4-nitrophenyl alpha-L-rhamnopyranoside + H2O
-
Novosphingobium sp. PP1Y 4-nitrophenol + alpha-L-rhamnopyranose
-
?
3.2.1.40 hesperidin + H2O
-
Novosphingobium sp. PP1Y hesperetin 7-O-beta-D-glucoside + alpha-L-rhamnose
-
?
3.2.1.40 additional information the enzyme is able to hydrolyze rhamnose from natural flavonoids. It hydrolyzes both alpha-1,2 and alpha-1,6 glycosidic linkages Novosphingobium sp. PP1Y ?
-
?
3.2.1.40 naringin + H2O
-
Novosphingobium sp. PP1Y beta-D-glucopyranosyl-2,3-dihydro-4',5,7-trihydroxyflavone + alpha-L-rhamnose
-
?
3.2.1.40 quercitrin + H2O
-
Novosphingobium sp. PP1Y quercetin + alpha-L-rhamnose
-
?
3.2.1.40 rutin + H2O
-
Novosphingobium sp. PP1Y isoquercitrin + alpha-L-rhamnose
-
?

Subunits

EC Number Subunits Comment Organism
3.2.1.40 monomer
-
Novosphingobium sp. PP1Y

Synonyms

EC Number Synonyms Comment Organism
3.2.1.40 GH106 alpha-L-rhamnosidase
-
Novosphingobium sp. PP1Y
3.2.1.40 RHA-P
-
Novosphingobium sp. PP1Y

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.40 35 45
-
Novosphingobium sp. PP1Y

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.40 12.2
-
quercitrin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 12.7
-
hesperidin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 18.2
-
rutin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 126
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, mutant enzyme D763A Novosphingobium sp. PP1Y
3.2.1.40 214
-
naringin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 458
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, mutant enzyme D552A Novosphingobium sp. PP1Y
3.2.1.40 692
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, wild-type enzyme Novosphingobium sp. PP1Y

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.40 6 7.5
-
Novosphingobium sp. PP1Y

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.1.40 80
-
quercitrin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 190
-
hesperidin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 360
-
naringin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 586
-
rutin pH 6.9, 37°C Novosphingobium sp. PP1Y
3.2.1.40 900
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, mutant enzyme D763A Novosphingobium sp. PP1Y
3.2.1.40 2900
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, mutant enzyme D552A Novosphingobium sp. PP1Y
3.2.1.40 4400
-
4-nitrophenyl alpha-L-rhamnopyranoside pH 6.9, 37°C, wild-type enzyme Novosphingobium sp. PP1Y