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 β-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.enoyl-CoA hydratase | epoxide | oxepin | oxygenase | phenylacetic acid T he biggest challenge for organisms using aromatic compounds as growth substrates is to overcome the stabilizing resonance energy of the aromatic ring system. This aromatic structure makes the substrates unreactive toward oxidation or reduction and thus requires elaborate degradation strategies. How microorganisms cope with this problem depends primarily on the availability of oxygen (1). Aerobic pathways use oxygen both for hydroxylation and for cleavage of the ring (2, 3). In contrast, under anaerobic conditions the common strategy consists of activation by CoA-thioester formation, shortening of the side chain, and energy-driven ring reduction, which also applies to phenylacetate catabolism (ref. 4 and literature cited therein).The aerobic strategy is illustrated by the metabolism of phenylacetate and phenylacetyl-CoA, which are derived from a variety of substrates such as phenylalanine, lignin-related phenylpropane units, 2-phenylethylamine, phenylalkanoic acids with an even number of carbon atoms, or even environmental contaminants such as styrene and ethylbenzene (5-7). Rarely, phenylalanine is hydroxylated to tyrosine, which can be converted into 4-hydroxyphenylpyruvate, followed by hydroxylation to homogentisate (2,5-dihydroxyphenylacetate) as the central intermediate. The aromatic ring of homogentisate then is split by a ring-cleaving homogentisate dioxygenase, and finally fumarate and acetoacetate are produced (8). In most cases, however, phenylalanine is converted into phenylacetate. A conventional aero...

Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization.

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The widespread, long sought-after bacterial aerobic phenylalanine/phenylacetate catabolic pathway has recently been elucidated. It proceeds via coenzyme A (CoA) thioesters and involves the epoxidation of the aromatic ring of phenylacetylCoA, subsequent isomerization to an uncommon seven-membered C-O-heterocycle (oxepin-CoA), and non-oxygenolytic ring cleavage. Here we characterize the hydrolytic oxepin-CoA ring cleavage catalyzed by the bifunctional fusion protein PaaZ. The enzyme consists of a C-terminal (R)-specific enoyl-CoA hydratase domain (formerly MaoC) that cleaves the ring and produces a highly reactive aldehyde and an N-terminal NADP ؉ -dependent aldehyde dehydrogenase domain that oxidizes the aldehyde to 3-oxo-5,6-dehydrosuberyl-CoA. In many phenylacetate-utilizing bacteria, the genes for the pathway exist in a cluster that contains an NAD ؉ -dependent aldehyde dehydrogenase in place of PaaZ, whereas the aldehyde-producing hydratase is encoded outside of the cluster. If not oxidized immediately, the reactive aldehyde condenses intramolecularly to a stable cyclic derivative that is largely prevented by PaaZ fusion in vivo. Interestingly, the derivative likely serves as the starting material for the synthesis of antibiotics (e.g. tropodithietic acid) and other tropone/tropolone related compounds as well as for -cycloheptyl fatty acids. Apparently, bacteria made a virtue out of the necessity of disposing the dead-end product with ring hydrolysis as a metabolic branching point.Aromatics like phenylacetic acid constitute the second most abundant class of natural organic compounds that serve as substrates mainly for microorganisms. Oxygen availability is key to how bacteria utilize such inert substrates (1). Under aerobic conditions oxygen is used to hydroxylate and cleave the ring (2, 3). In contrast, under anaerobic conditions the inert substrates become activated to CoA-thioesters followed by shortening of the side chain and energy-driven ring reduction; furthermore, ring cleavage occurs hydrolytically rather than by oxygenation. This strategy also applies to anaerobic phenylacetate catabolism (Ref. 4 and literature cited therein) (Fig. 1A).Phenylacetate (I) is a key intermediate in the degradation of various substrates like phenylalanine, lignin-related aromatic compounds, or environmental contaminants (5, 6). The first studies from more than 20 years ago reported the induction of a phenylacetate-CoA ligase under aerobic conditions in Pseudomonas putida, suggesting an unconventional strategy for aerobic phenylacetic acid (Paa) 2 degradation (7, 8). A total of 14 genes in 3 transcriptional units were identified in the corresponding paa gene cluster (9). However, the underlying biochemical conversions remained obscure until they were recently elucidated in Escherichia coli K12 and Pseudomonas sp. strain Y2 (10). This novel metabolic strategy involves the usage of oxygen as well as CoA-thioester intermediates throughout the pathway, hence, showing typical features of aerobic as well as anaerobic strategies...