Engleder M et al. (2018), Recombinant expression, purification and bioche...

ECB-ART-46122

PLoS One 2018 Feb 08;132:e0192653. doi: 10.1371/journal.pone.0192653.

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Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca.

Engleder M , Horvat M , Emmerstorfer-Augustin A , Wriessnegger T , Gabriel S , Strohmeier G , Weber H , Müller M , Kaluzna I , Mink D , Schürmann M , Pichler H .

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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. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F. solani, based on in silico sequence analyses. After heterologous expression of the enzyme in the methylotrophic yeast Pichia pastoris, we have confirmed its kievitone hydration activity and have assessed its biochemical properties and substrate specificity. Purified recombinant NhKHS is obviously a homodimeric glycoprotein. Due to its good activity for the readily available chalcone derivative xanthohumol (XN), this compound was selected as a model substrate for biochemical studies. The optimal pH and temperature for hydratase activity were 6.0 and 35°C, respectively, and apparent Vmax and Km values for hydration of XN were 7.16 μmol min-1 mg-1 and 0.98 ± 0.13 mM, respectively. Due to its catalytic properties and apparent substrate promiscuity, NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols.

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Genes referenced: LOC115919910 LOC577676

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	Fig 1. Analysis of secreted protein and purification of NhKHS from P. pastoris culture supernatant (A). Proteins were precipitated from the culture supernatant of P. pastoris wild type strain (lane 1) and PpKHSAlpha strain (lane 2) with the chloroform/methanol method. Four hundred μL of cell culture were precipitated. KHS was purified via Ni-NTA affinity chromatography. Ten μL of flow through (lane 4), washing fractions with 10 mM imidazole (lanes 5 and 6) and 50 mM imidazole (lanes 7 and 8), as well as 4 μL of concentrated, pooled elution fractions (lane 9) were loaded onto the gel. PageRulerTM Prestained Protein Ladder was used as molecular weight standard (Lane 3). In vitro deglycosylation of purified KHS with EndoHf (B). Deglycosylation reactions were incubated for 0.5–2.5 h.

	Fig 3. Flavonoid substrates converted with NhKHS.

	Fig 5. Kinetic characterization of NhKHS with the substrate XN.Reaction mixtures contained 0.05 mg mL-1 of purified enzyme in 50 mM sodium citrate, pH 6.0, with 2% ethanol (v/v) and the XN concentration was varied from 0.125 to 3.0 mM. Reactions were incubated for 2 min at 35°C and 150 rpm in triplicates. (A) Michaelis-Menten plot. Kinetic parameters were determined directly from the regression data of a nonlinear hyperbolic curve fit. (B) Summary of kinetic constants for XN hydration by NhKHS.
	Fig 6. Effect of reaction conditions on the conversion of XN by NhKHS.For determination of reaction optima, 100 μg of purified enzyme were incubated with 0.5 mM substrate for 10 min at 28°C in triplicates. (A) The effect of pH on NhKHS activity was investigated in 50 mM sodium citrate at pH 4.5–6.0, 50 mM potassium phosphate at pH 7.0–8.0 and 50 mM Tris-HCl at pH 9.0. (B) The effect of temperature on enzyme activity was determined by incubation of reactions at 15°C- 40°C.