Characterization of p-hydroxycinnamate catabolism in a soil Actinobacterium

Abstract

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 upregulated during growth on ferulate versus benzoate. The upregulated genes included three 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 (k(cat)/K(m) = ∼ 400 mM(-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 two p-coumaric acid:coenzyme A (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. The results of this study augment our understanding of the bacterial catabolism of aromatics from renewable feedstocks.

FIG 1

Structures of the aromatic compounds used in this study. 1, p-coumarate; 2, ferulate; 3, caffeate; 4, dihydroferulate; 5, dihydro-p-coumarate; 6, sinapate; 7, dihydrocoumarin; 8, eugenol; 9, benzoate; 10, vanillate. Compounds 1 to 6 are p-hydroxycinnamates.

FIG 2

(A) The cou gene cluster. Black arrows indicate genes highly transcribed in ferulate-grown cells. (B) The Cou pathway of RHA1. Ferulate (R = OCH₃) is activated to feruloyl-CoA by CouL. Feruloyl-CoA is hydrated to 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA and then oxidized to 4H3MPKP-CoA in reactions catalyzed by CouM and CouN, respectively. Finally, CouO catalyzes the hydrolysis of 4H3MPKP-CoA to vanillate. Formaldehyde is formed by the demethylation of vanillate to protocatechuate (see Fig. S1 in the supplemental material). p-Coumarate (R = H) is catabolized in the same manner, forming 4-hydroxybenzoate. 4H3MPKP-CoA and 4HPKP-CoA may be hydrolyzed if they accumulate intracellularly, resulting in 4H3MPKP and 4HPKP, respectively, which are then excreted. (C) Mycothiol-dependent formaldehyde detoxification gene cluster. (D) RT-PCR analysis of the couHTL genes. Total RNA used to synthesize the cDNA was isolated from the RHA1 cells grown on ferulate. Positions of the amplicons are indicated in panel B. G, genomic DNA; +, reverse transcriptase positive; –, reverse transcriptase negative.

FIG 3

HPLC analysis of p-hydroxycinnamate-derived metabolites. (A) Culture supernatants of the wt strain and the ΔcouL and ΔcouO mutants after 0 and 2 days of incubation with ferulate. Peak 1 corresponds to ferulate. Peaks 2 (m/z = 211.06) and 3 (m/z = 167.07) were identified as protonated 4-hydroxy-3-methoxyphenyl-β-ketopropionate and acetovanillone, respectively. (B) The corresponding experiment after 0 and 1 day of incubation with p-coumarate. Peak 1 corresponds to p-coumarate. Peaks 2 (m/z = 181.05) and 3 (m/z = 137.06) were identified as protonated 4-hydroxyphenyl-β-ketopropionate and 4′-hydroxyacetophenone, respectively.

FIG 4

CouL is a p-hydroxycinnamoyl-CoA synthetase. Data represent the results of reverse-phase HPLC analysis of CouL reaction with ferulate (A), p-coumarate (B), caffeate (C), or dihydroferulate (D). ATP, CoASH, and p-hydroxycinnamate were incubated with (red line) or without (black line) CouL at 30°C and analyzed by reverse-phase HPLC. Peak 1 corresponds to p-hydroxycinnamate. The insets show the MS spectra of peak 2 analyzed by LC-ESI-MS. (A) The peak of the inset at the m/z of 944.0 corresponds to protonated feruloyl-CoA. The peak at the m/z of 921.9 was also observed in a blank sample. (B) The peak of the inset at the m/z of 914.1 corresponds to protonated p-coumaroyl-CoA. The peak at the m/z of 921.9 was also observed in a blank sample. (C) The peak of the inset at the m/z of 930.0 corresponds to protonated caffeoyl-CoA. The peak at the m/z of 921.9 was also observed in a blank sample. (D) The peak of the inset at the m/z of 946.1 corresponds to protonated dihydroferuloyl-CoA. The peaks at the m/z of 921.9 and 913.9 were also observed in a blank sample.

FIG 5

Steady-state kinetics of CouL-catalyzed thioesterification of p-hydroxy-cinnamates. Reactions were performed using 1 mM ATP, 2 mM CoASH, 10 mM MgCl₂, and 0.1 M potassium phosphate (pH 7.0) at 30°C. The solid line represents the best fits of the Michaelis-Menten equation to the data using LEONORA.