Abstract
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.
Highlights
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]
Several studies have shown that detoxification of the plant isoflavonoid phytoalexins KV and phaseollidin by F. solani f. sp. phaseoli is conferred by at least two different enzymatic
Kievitone hydratase activity was detected for other members of the F. solani species complex, such as F. oxysporum and N. haematococca by showing production of hydroxy– kievitone (HO-KV) from KV upon infection of the French bean [16]
Summary
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]. After identifying a putative kievitone hydratase sequence in N. haematococca MP VI, we have heterologously expressed the gene in P. pastoris and confirmed its activity for KV and other flavonoids. The activity of the NhKHS protein was determined by applying in vitro assays with either culture supernatant containing secreted NhKHS or His10-tag purified NhKHS.
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