Characterization of p-Hydroxycinnamate Catabolism in a Soil Actinobacterium.

p-Hydroxycinnamates, such as ferulate and p-coumarate, are components of plant cell walls and have a number of commercial applications. Rhodococcus jostii RHA1 (RHA1) catabolizes ferulate via vanillate and the β-ketoadipate pathway. Here, we used transcriptomics to identify genes in RHA1 that are up-regulated during growth on ferulate versus benzoate. The up-regulated genes included three transcriptional units predicted to encode the uptake and β-oxidative deacetylation of p-hydroxycinnamates: couHTL, couNOM and couR. Neither ΔcouL nor ΔcouO mutants grew on p-hydroxycinnamates, but grew on vanillate. Among several p-hydroxycinnamates, CouL catalyzed the thioesterification of p-coumarate and caffeate most efficiently (kcat/Km = ~400 mM-1s-1). p-Coumarate was also RHA1's preferred growth substrate, suggesting that CouL is a determinant of the pathway's specificity. CouL did not catalyze the activation of sinapate, similar to two p-coumaric acid:CoA ligases from plants, and contains the same bulged loop that helps determine substrate specificity in the plant homologues. The couO mutant accumulated 4-hydroxy-3-methoxyphenyl-β-ketopropionate in the culture supernatant when incubated with ferulate, supporting β-oxidative deacetylation. This phenotype was not complemented with a D257N variant of CouO, consistent with the predicted role of Asp257 as a metal ligand in this amidohydrolase superfamily member. These data suggest that CouO functionally replaces the β-ketothiolase and acyl-CoA thioesterase that occur in canonical β-oxidative pathways. Finally, the transcriptomics data suggest the involvement of two distinct formaldehyde detoxification pathways in vanillate catabolism and identify a eugenol catabolic pathway. This study augments our understanding of the bacterial catabolism of aromatics from renewable feedstocks. [ABSTRACT FROM AUTHOR]