Research Article: Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca

Date Published: February 8, 2018

Publisher: Public Library of Science

Author(s): Matthias Engleder, Melissa Horvat, Anita Emmerstorfer-Augustin, Tamara Wriessnegger, Stefanie Gabriel, Gernot Strohmeier, Hansjörg Weber, Monika Müller, Iwona Kaluzna, Daniel Mink, Martin Schürmann, Harald Pichler, Israel Silman.


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.

Partial Text

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 [1,2]. However, synthesis of tertiary alcohols is still a demanding task for synthetic organic chemistry due to issues such as low yields, poor selectivity or harsh reaction conditions. Therefore, sustainable biocatalytic processes that rely on enzymatic transformations are highly desirable [2,3]. The most extensively applied enzymes for synthesis of optically active, tertiary alcohols belong to the class of hydrolases [4,5], where especially lipases and esterases have been used for their kinetic resolution [1,2]. In contrast, members of other enzyme classes are markedly underrepresented, even given the fact that some of them would offer great possibilities for applications. This holds particularly true for hydro-lyases (EC 4.2.1.X), 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 [6,7]. Aside from cofactor dependent hydro-lyases, a number of enzymes catalyze the addition of water cofactor-independently, which increases the potential of this enzyme group for industrial applications. However, although more than 100 hydro-lyases have been discovered to date, only a very limited number has been applied industrially. The most prominent examples include nitrile hydratase and fumarase for the production of acrylamide on a 30,000 t a-1 scale, or the production of (S)-malic acid (2,500 t a-1), respectively [7]. Another enzyme group that has recently received increasing attention of both academic and industrial research are oleate hydratases. Representatives of this class of hydratases have been applied for the production of 10-hydroxystearic acid from oleic acid, as well as α,ω-dicarboxylic acids, ω-hydroxycarboxylic acids and γ-dodecalactones in multistep enzymatic reaction systems [8–10]. Compounds obtained from oleate hydratase reactions are applied widespread in the production of a large variety of chemicals and intermediates, such as polymers, coatings, lubricants, personal care, perfumes and food additives [11].




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