Proteogenomic evidence for β-oxidation of plant-derived 3-phenylpropanoids in Aromatoleum aromaticum EbN1

The betaproteobacterium Aromatoleum aromaticum EbN1 utilizes eight different plant-derived nonhydroxylated (e. g. cinnamate) and hydroxylated (e. g. p-coumarate) 3-phenylpropanoids with nitrate as electron acceptor. Differential protein profiling (2D-DIGE) revealed abundance increases of five proteins (EbA5316 to EbA5320) during anaerobic growth with cinnamate, hydrocinnamate, p-coumarate, and 3-(4-hydroxyphenyl) propanoate, compared to anaerobic benzoate-adapted cells serving as reference state. The predicted functions of four of these proteins (EbA5317, fatty acid-coenzyme A (CoA) ligase; EbA5318, enoyl-CoA hydratase/isomerase; EbA5319, beta-ketothiolase; and EbA5320, 3-hydroxyacyl-CoA dehydrogenase) suggest beta-oxidation of the above 3-phenylpropanoids to benzoyl-CoA and p-hydroxybenzoyl-CoA, respectively. The fifth protein (EbA5316, ABC-type periplasmic solute-binding protein) could be involved in 3-phenylpropanoid uptake. The detection of 3-hydroxy-3-phenylpropanoate during anaerobic growth with cinnamate and hydrocinnamate or 3-hydroxy-3-(4-hydroxyphenyl) propanoate during anaerobic growth with p-coumarate and 3-(4-hydroxyphenyl) propanoate supports the proteome-predicted p-oxidation pathway. Based on the specific formation of EbA5316-20 also during anaerobic growth with further 3-phenylpropanoid growth substrates including cinnamyl alcohol, m-coumarate, 3-(3,4-dihydroxyphenyl) propanoate and 3,4-dihydroxycinnamate (caffeate), a common beta-oxidation route is proposed for 3-phenylpropanoid degradation in strain EbN1. The low amount of metabolites attributable to cometabolic transformation of nongrowth supporting 3-phenylpropanoids (e. g. o-coumarate, ferulate) may be indicative for a high substrate specificity of the involved enzymes.