Unravelling the Reduction Pathway as an Alternative Metabolic Route to Hydroxycinnamate Decarboxylation in Lactobacillus plantarum

Abstract

is the lactic acid bacterial species most frequently found in plant-food fermentations where hydroxycinnamic acids are abundant. efficiently decarboxylates these compounds and also reduces them, yielding substituted phenylpropionic acids. Although the reduction step is known to be induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this reduction pathway has not been yet identified and characterized. A previous study on the transcriptomic response of to -coumaric acid revealed a marked induction of two contiguous genes, and , encoding putative reductases. In this work, the disruption of these genes abolished the hydroxycinnamate reductase activity of, supporting their involvement in such chemical activity. Functional studies revealed that Lp_1425 (HcrB) exhibits hydroxycinnamate reductase activity but was unstable in solution. In contrast, Lp_1424 (HcrA) was inactive but showed high stability. When the genes were co-overexpressed, the formation of an active heterodimer (HcrAB) was observed. Since reductase activity was only observed on hydroxycinnamic acids (-coumaric, -coumaric,-coumaric, caffeic, ferulic, and sinapic acids), the presence of a hydroxyl group substituent on the benzene ring appears to be required for activity. In addition, hydroxycinnamate reductase activity was not widely present among lactic acid bacteria, and it was associated with the presence of genes. This study revealed that hydroxycinnamate reductase is a heterodimeric NADH-dependent coumarate reductase acting on a carbon-carbon double bond. is a bacterial species frequently found in the fermentation of vegetables where hydroxycinnamic acids are present. The bacterial metabolism on these compounds during fermentation plays a fundamental role in the biological activity of hydroxycinnamates. strains exhibit an as yet unknown reducing activity, transforming hydroxycinnamates to substituted phenylpropionic acids, which possess higher antioxidant activity than their precursors. The protein machinery involved in hydroxycinnamate reduction, HcrAB, was genetically identified and characterized. The heterodimeric NADH-dependent coumarate reductase HcrAB described in this work provides new insights on the metabolic response to counteract the stressful conditions generated by food phenolics.

Ethylphenol Formation by Lactobacillus plantarum: Identification of the Enzyme Involved in the Reduction of Vinylphenols

“…Since the reaction catalyzed by HcrAB also implies the reduction of a carbon-carbon double bond, the possible involvement of HcrAB in vinylphenol reduction needs to be eliminated. To this end, the HrcA and HcrB knockout mutants (28) were grown in the presence of 4-vinylphenol and, as shown in Fig. 1, both mutants were able to reduce this compound, demonstrating that neither of the two proteins was involved in vinylphenol reduction.…”

“…As expected, the L. plantarum transcriptomic response to p-coumaric acid revealed the induction of the pdc gene (112-fold), but also of the genes encoding hydroxycinnamate reductase (hcrA and hcrB; 15.3 and 19.7-fold, respectively). In this regard, we have recently described the involvement of HcrAB in the reduction of hydroxycinnamic acids into substituted phenylpropionic acid (28). Since the reaction catalyzed by HcrAB also implies the reduction of a carbon-carbon double bond, the possible involvement of HcrAB in vinylphenol reduction needs to be eliminated.…”

“…6). Some of these acids could be reduced to their corresponding substituted phenylpropionic acids by the action of recently described HcrAB proteins (encoded by lp_1424 and lp_1425 genes) (28). m-Coumaric, o-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids are also reduced by HcrAB to originate 3-(3hydroxyphenyl) propionic, melilotic, phloretic, hydrocaffeic, hydroferulic, and hydrosinapic acids, respectively (28).…”

“…L. plantarum WCFS1 MRS cultures were grown to an optical density at 600 nm (OD 600 ) of 0.8 to 0.9 and then supplemented with 4-vinylphenol or p-coumaric acid at 1.5 mM final concentration. After 10 min of incubation, the cultures were immediately processed for RNA extraction, and, after DNase I treatment, the DNA-free RNA was retrotranscribed (28). The cDNA obtained in the presence of 4-vinylphenol was used for RT-PCR experiments.…”

“…The lp_3123, vprR (lp_3124), vprA (lp_3125), and lp_3127 genes as well as the lp_3123-vprR, vprR-vprA, and vprA-lp_3127 intergenic regions were analyzed by PCR. PCR amplifications were performed as previously described (28).…”