Biochemical characterization of HcrF from Limosilactobacillus fermentum, a NADH-dependent 2-ene reductase with activity on hydroxycinnamic acids.

In fermented plant foods, phenolic compounds are metabolized by 2-ene reductases, which reduce double bonds adjacent to an aromatic rings in phytochemicals, including hydroxycinnamic acids, isoflavones, and flavones. Only few 2-ene reductases of lactic acid bacteria were characterized, including the hydrocinnamic reductases HcrB and Par1, and the daidzein reductase of Lactococcus lactis. This study aimed to characterize HcrF, a homologue of HcrB, in Limosilactobacillus fermentum. HcrF was purified after cloning in Escherichia coli and purification by affinity chromatography. HcrF was optimally active at 30°C-40°C and pH 7.0 and required both flavin mononucleotide and nicotinamide adenine dinucleotide as co-factors. Ferulic, caffeic, p-coumaric, and sinapic acids but not trans-cinnamic acids were reduced to dihydro derivatives. The maximum reaction velocity Vmax of HcrF was highest for ferulic acid. On a phylogenetic tree of 2-ene reductases, HcrF clustered most closely with the hydroxycinnamic acid reductase HcrB of Lactiplantibacillus plantarum. The hydroxycinnamic acid reductase Par1 of Furfurilactobacillus milii and flavone or isoflavone reductases were only distantly related to HcrF. In summary, current knowledge does not allow to predict the substrate specificity of 2-ene reductases on the basis of the protein sequence; this study adds HcrF to the short list of enzymes with known substrate specificity.