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Literature summary extracted from

  • Engleder, M.; Horvat, M.; Emmerstorfer-Augustin, A.; Wriessnegger, T.; Gabriel, S.; Strohmeier, G.; Weber, H.; Mueller, M.; Kaluzna, I.; Mink, D.; Schuermann, M.; Pichler, H.
    Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca (2018), PLoS ONE, 13, e0192653 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
4.2.1.95 synthesis due to its catalytic properties and apparent substrate promiscuity, enzyme NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols. The production of enantiopure tertiary alcohols is a major challenge of organic synthesis as these functional groups are widely applicable for the generation of pharmaceuticals or other bioactive compounds Fusarium solani
4.2.1.95 synthesis due to its catalytic properties and apparent substrate promiscuity, enzyme NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols. The production of enantiopure tertiary alcohols is a major challenge of organic synthesis as these functional groups are widely applicable for the generation of pharmaceuticals or other bioactive compounds. The compatibility of enzyme NhKHS with nonpolar organic solvents is beneficial for a number of biocatalytic applications, e.g. in general for conversion of hydrophobic substrates Fusarium solani

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.95 DNA and amino acid sequence determination and analysis, sequence comparisons, higher functional recombinant expression of codon-harmonized gene encoding the enzyme in Pichia pastoris (i.e. Komagataella phaffii) strain CBS7435 as C-terminally His10-tagged protein, the mature enzyme is secreted. Upscaling tests show that Pichia pastoris is a most appropriate host for producing high amounts of recombinant NhKHS Fusarium solani
4.2.1.95 DNA and amino acid sequence determination and analysis, sequence comparisons, lower functional recombinant expression of codon-harmonized gene encoding the enzyme in Pichia pastoris (i.e. Komagataella phaffii) strain CBS7435 as C-terminally His10-tagged protein Fusarium solani

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.1.95 additional information
-
additional information Michaelis-Menten kinetics Fusarium solani
4.2.1.95 0.98
-
xanthohumol pH 6.0, 35°C, recombinant enzyme Fusarium solani

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
4.2.1.95 additional information the enzyme contains a 19 amino acid-long N-terminal signal peptide whose cleavage site is predicted between alanine 19 and lysine 20. Absence of nucleotide cofactors is assumed due to the absence of a conserved nucleotide binding motif Fusarium solani
-
-
4.2.1.95 additional information the enzyme contains a 19 amino acid-long N-terminal signal peptide whose cleavage site is predicted between alanine 19 and serine 20. The signal peptide is absent from the predominantly secreted mature enzyme Fusarium solani
-
-

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.2.1.95 75000
-
recombinant His-tagged mature and glycosylated enzyme, gel filtration Fusarium solani

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.1.95 kievitone hydrate Fusarium solani
-
kievitone + H2O
-
r
4.2.1.95 kievitone hydrate Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI
-
kievitone + H2O
-
r

Organic Solvent Stability

EC Number Organic Solvent Comment Organism
4.2.1.95 chloroform does affect the activity up to 5%, slight inhibition of xanthohumol hydrate formation at 10%, moderate inhibition at 30% Fusarium solani
4.2.1.95 DMSO slight inhibition of xanthohumol hydrate formation at 5%, higher inhibition at 10%, complete inhibition at 30% Fusarium solani
4.2.1.95 dodecane does affect the activity up to 30% Fusarium solani
4.2.1.95 Ethanol does affect the activity up to 5%, moderate inhibition of xanthohumol hydrate formation at 10%, strong inhibition at 30% Fusarium solani
4.2.1.95 hexane does affect the activity up to 25%, slight inhibition of xanthohumol hydrate formation at 30% Fusarium solani
4.2.1.95 additional information the polarity of the organic solvent is a determining factor for its effect on NhKHS activity, with increasing solubility in water resulting in a more distinct impact. This might be caused by an enhanced perturbing effect on the hydration shell of the enzyme Fusarium solani

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.95 Fusarium solani C7ZKN4 i.e. Fusarium solani subsp. pisi
-
4.2.1.95 Fusarium solani Q00870 i.e. Fusarium solani subsp. phaseoli
-
4.2.1.95 Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI C7ZKN4 i.e. Fusarium solani subsp. pisi
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
4.2.1.95 glycoprotein N-glycosylation Fusarium solani
4.2.1.95 glycoprotein the enzyme contains four typical consensus sequences for N-linked glycosylation. The temperature stability of the enzyme is apparently not affected by deglycosylation in vivo, while in vitro activity assays show that the activity of the deglycosylated enzyme is approx. 30% lower compared to the glycosylated control Fusarium solani

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.95 recombinant His10-tagged enzyme from Pichia pastoris strain CBS7435 by nickel affinity chromatography and gel filtration Fusarium solani

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.95 8-prenylnaringenin + H2O low activity Fusarium solani 8-prenylnaringenin hydrate
-
r
4.2.1.95 8-prenylnaringenin + H2O low activity Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI 8-prenylnaringenin hydrate
-
r
4.2.1.95 8-prenylnaringenin hydrate
-
Fusarium solani 8-prenylnaringenin + H2O
-
r
4.2.1.95 8-prenylnaringenin hydrate
-
Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI 8-prenylnaringenin + H2O
-
r
4.2.1.95 isoxanthohumol + H2O
-
Fusarium solani isoxanthohumol hydrate
-
r
4.2.1.95 isoxanthohumol hydrate
-
Fusarium solani isoxanthohumol + H2O
-
r
4.2.1.95 kievitone + H2O best substrate Fusarium solani kievitone hydrate
-
r
4.2.1.95 kievitone + H2O best substrate Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI kievitone hydrate
-
r
4.2.1.95 kievitone hydrate
-
Fusarium solani kievitone + H2O
-
r
4.2.1.95 kievitone hydrate
-
Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI kievitone + H2O
-
r
4.2.1.95 additional information substrate specificity analysis of the heterologously expressed recombinant enzyme, confirmation of HO-XN formation from XN by 1H-NMR and 13C-NMR in vitro Fusarium solani ?
-
?
4.2.1.95 additional information substrate specificity analysis of the heterologously expressed recombinant enzyme, confirmation of HO-XN formation from XN by 1H-NMR and 13C-NMR in vitro Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI ?
-
?
4.2.1.95 xanthohumol XN, high activity with the recombinant enzyme Fusarium solani ?
-
r
4.2.1.95 xanthohumol + H2O XN, high activity with the recombinant enzyme Fusarium solani xanthohumol hydrate
-
r
4.2.1.95 xanthohumol hydrate
-
Fusarium solani xanthohumol + H2O
-
r

Subunits

EC Number Subunits Comment Organism
4.2.1.95 homodimer
-
Fusarium solani
4.2.1.95 homodimer 2 * 48000, recombinant His-tagged mature and glycosylated enzyme, SDS-PAGE Fusarium solani

Synonyms

EC Number Synonyms Comment Organism
4.2.1.95 FsKHS
-
Fusarium solani
4.2.1.95 NhKHS
-
Fusarium solani

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.1.95 35
-
recombinant enzyme Fusarium solani

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
4.2.1.95 additional information
-
the temperature stability of the enzyme is apparently not affected by deglycosylation in vivo, while in vitro activity assays show that the activity of the deglycosylated enzyme is approx. 30% lower compared to the glycosylated control Fusarium solani

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.1.95 4.77
-
xanthohumol pH 6.0, 35°C, recombinant enzyme Fusarium solani

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.95 6
-
recombinant enzyme Fusarium solani

pH Range

EC Number pH Minimum pH Maximum Comment Organism
4.2.1.95 4 8 activity range, recombinant enzyme, profile overview Fusarium solani

General Information

EC Number General Information Comment Organism
4.2.1.95 evolution the enzyme belongs to the hydro-lyases, which are able to catalyze the highly selective, reversible addition of water to non-activated carbon-carbon double bonds and, thereby, generate primary, secondary or tertiary alcohols Fusarium solani
4.2.1.95 evolution the enzyme is a member of the hydrolyase enzyme group Fusarium solani
4.2.1.95 evolution the enzyme is a member of the hydrolyase enzyme group. Hydro-lyases are able to catalyze the highly selective, reversible addition of water to non-activated carbon-carbon double bonds and, thereby, generate primary, secondary or tertiary alcohols Fusarium solani
4.2.1.95 physiological function kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection Fusarium solani

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.2.1.95 4.88
-
xanthohumol pH 6.0, 35°C, recombinant enzyme Fusarium solani