Bacterial phenylalanine and phenylacetate catabolic pathway revealed

Abstract

Aromatic compounds constitute the second most abundant class of organic substrates and environmental pollutants, a substantial part of which (e.g., phenylalanine or styrene) is metabolized by bacteria via phenylacetate. Surprisingly, the bacterial catabolism of phenylalanine and phenylacetate remained an unsolved problem. Although a phenylacetate metabolic gene cluster had been identified, the underlying biochemistry remained largely unknown. Here we elucidate the catabolic pathway functioning in 16% of all bacteria whose genome has been sequenced, including Escherichia coli and Pseudomonas putida. This strategy is exceptional in several aspects. Intermediates are processed as CoA thioesters, and the aromatic ring of phenylacetyl-CoA becomes activated to a ring 1,2-epoxide by a distinct multicomponent oxygenase. The reactive nonaromatic epoxide is isomerized to a seven-member O-heterocyclic enol ether, an oxepin. This isomerization is followed by hydrolytic ring cleavage and beta-oxidation steps, leading to acetyl-CoA and succinyl-CoA. This widespread paradigm differs significantly from the established chemistry of aerobic aromatic catabolism, thus widening our view of how organisms exploit such inert substrates. It provides insight into the natural remediation of man-made environmental contaminants such as styrene. Furthermore, this pathway occurs in various pathogens, where its reactive early intermediates may contribute to virulence.

Fig. 1.

Aerobic phenylacetate catabolic pathway. (A) Catabolic gene cluster for phenylacetate degradation in E. coli K12. (B) Reactions and intermediates of the pathway as studied in E. coli K12 and Pseudomonas sp. strain Y2. Proposed enzyme names (Table S1): 1: phenylacetate-CoA ligase (AMP forming); 2: ring 1,2-phenylacetyl-CoA epoxidase (NADPH); 3: ring 1,2-epoxyphenylacetyl-CoA isomerase (oxepin-CoA forming), postulated 3,4-dehydroadipyl-CoA isomerase. 4: oxepin-CoA hydrolase/ 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase (NADP⁺); 5: 3-oxoadipyl-CoA/ 3-oxo-5,6-dehydrosuberyl-CoA thiolase; 6: 2,3-dehydroadipyl-CoA hydratase; 7: 3-hydroxyadipyl-CoA dehydrogenase (NAD⁺) (probably (S)-3-specific). Compounds: I, phenylacetate; II, phenylacetyl-CoA; III, ring 1,2-epoxyphenylacetyl-CoA; IV, 2-oxepin-2(3H)-ylideneacetyl-CoA; V, 3-oxo-5,6-dehydrosuberyl-CoA, VI, 2,3-dehydroadipyl-CoA; VII, acetyl-CoA; VIII, 3-hydroxyadipyl-CoA; IX, 3-oxoadipyl-CoA; X, succinyl-CoA.

Fig. 2.

Conversion of phenylacetyl-CoA to succinyl-CoA and acetyl-CoA at 30 °C by heterologously produced enzymes of the phenylacetate pathway. The initial reaction mixture (0.25 mL) contained 50 mM Tris-HCl (pH 8.0), 0.5 mM CoA, 1 mM NADPH, 1 mM NADP⁺, and 0.8 mg of PaaABC(D)E. The reaction was started by addition of 0.5 mM phenylacetyl-CoA. The products were separated by RP-HPLC and detected at 260 nm. (A) Control without PaaABC(D)E. (B) Three minutes after substrate addition (ring 1,2-epoxyphenylacetyl-CoA). (C) Three minutes after addition of 10 μg PaaG (oxepin-CoA). Because of an inactivation of the enzymes (likely through nonconverted epoxides), a second reaction mixture (0.4 mL) was used for the following samples. From the start of the reaction it contained PaaABC(D)E, as above, and 10 μg of PaaG and 30 μg of PaaZ. (D) After 2 min (3-oxo-5,6-dehydrosuberyl-CoA). (E) Four minutes after addition of 7 μg PaaJ (2,3-dehydroadipyl-CoA and acetyl-CoA, consumption of CoA). (F) Three minutes of incubation with 2 μg of PaaF (3-hydroxyadipyl-CoA). (G) Three minutes of incubation with 16 μg of PaaH, 0.5 mM CoA, and 1 mM NAD⁺ (succinyl-CoA and acetyl-CoA). The PaaH product 3-oxoadipyl-CoA is directly converted by PaaJ and therefore is not shown.

Fig. 3.

(A) Proposed PaaG-catalyzed ring expansion (oxepin-CoA–forming) through isomerization. Note that the numbering of ring C atoms changes through oxepin-CoA formation. (B) Proposed hydrolytic ring cleavage of oxepin-CoA and subsequent aldehyde oxidation by fusion protein PaaZ. The roman numerals indicate the compounds in Fig. 1B.