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 no. of com. applications. Rhodococcus jostii RHA1 catabolizes ferulate via vanillate and the β-ketoadipate pathway. Here, we used transcriptomics to identify genes in RHA1 that are upregulated during growth on ferulate vs. benzoate. The upregulated genes included 3 transcriptional units predicted to encode the uptake and β-oxidative deacetylation of p-hydroxycinnamates: couHTL, couNOM, and couR. Neither ΔcouL mutants nor ΔcouO mutants grew on p-hydroxycinnamates, but they did grow on vanillate. Among several p-hydroxycinnamates, CouL catalyzed the thioesterification of p-coumarate and caffeate most efficiently (kcat/Km = ∼400mM-1 s-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, in similarity to 2 p-coumaric acid:CoA ligases from plants, and contains the same bulged loop that helps det. substrate specificity in the plant homologs. 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 2 distinct formaldehyde detoxification pathways in vanillate catabolism and identify a eugenol catabolic pathway. The results of this study augment our understanding of the bacterial catabolism of aroms. from renewable feedstocks. [on SciFinder(R)]

J Bacteriol