Literature summary for 4.2.1.95 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 .

Application

Application	Comment	Organism
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
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)

Cloned (Comment)	Organism
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
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

Cloned (Comment)

Organism

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

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]

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

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
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	-	-
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]

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

Natural Substrates/ Products (Substrates)

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

Organic Solvent Stability

Organic Solvent	Comment	Organism
chloroform	does affect the activity up to 5%, slight inhibition of xanthohumol hydrate formation at 10%, moderate inhibition at 30%	Fusarium solani
DMSO	slight inhibition of xanthohumol hydrate formation at 5%, higher inhibition at 10%, complete inhibition at 30%	Fusarium solani
dodecane	does affect the activity up to 30%	Fusarium solani
Ethanol	does affect the activity up to 5%, moderate inhibition of xanthohumol hydrate formation at 10%, strong inhibition at 30%	Fusarium solani
hexane	does affect the activity up to 25%, slight inhibition of xanthohumol hydrate formation at 30%	Fusarium solani
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

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

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	N-glycosylation	Fusarium solani
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)

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
8-prenylnaringenin + H2O	low activity	Fusarium solani	8-prenylnaringenin hydrate	-	r
8-prenylnaringenin + H2O	low activity	Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI	8-prenylnaringenin hydrate	-	r
8-prenylnaringenin hydrate	-	Fusarium solani	8-prenylnaringenin + H2O	-	r
8-prenylnaringenin hydrate	-	Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI	8-prenylnaringenin + H2O	-	r
isoxanthohumol + H2O	-	Fusarium solani	isoxanthohumol hydrate	-	r
isoxanthohumol hydrate	-	Fusarium solani	isoxanthohumol + H2O	-	r
kievitone + H2O	best substrate	Fusarium solani	kievitone hydrate	-	r
kievitone + H2O	best substrate	Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI	kievitone hydrate	-	r
kievitone hydrate	-	Fusarium solani	kievitone + H2O	-	r
kievitone hydrate	-	Fusarium solani 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI	kievitone + H2O	-	r
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	?	-	?
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	?	-	?
xanthohumol	XN, high activity with the recombinant enzyme	Fusarium solani	?	-	r
xanthohumol + H2O	XN, high activity with the recombinant enzyme	Fusarium solani	xanthohumol hydrate	-	r
xanthohumol hydrate	-	Fusarium solani	xanthohumol + H2O	-	r

Subunits

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

Synonyms

Synonyms	Comment	Organism
FsKHS	-	Fusarium solani
NhKHS	-	Fusarium solani

Temperature Optimum [°C]

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

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
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]

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

pH Optimum

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

pH Range

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

General Information

General Information	Comment	Organism
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
evolution	the enzyme is a member of the hydrolyase enzyme group	Fusarium solani
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
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]

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