Anaerobic Metabolism of Aromatic Compounds

Anaerobic Metabolism of Aromatic Compounds GEORG FUCHS Microbiology, Faculty of Biology, University of Freiburg, Freiburg, Germany Aromatic compounds comprise a wide variety of natural and synthetic compounds that can serve as substrates for bacterial growth. So far, four types of aromatic metabolism are known. (1) The aerobic aromatic metabolism is characterized by the extensive use of molecular oxygen as cosubstrate for oxygenases that introduce hydroxyl groups and cleave the aromatic ring. (2) In the presence of oxygen, facultative aerobes use another so-called hybrid type of aerobic metabolism of benzoate, phenylacetate, and anthranilate (2-aminobenzoate). These pathways use coenzyme A thioesters of the substrates and do not require oxygen for ring cleavage; rather they use an oxygenase/reductase to dearomatize the ring. (3) In the absence of oxygen, facultative aerobes and phototrophs use a reductive aromatic metabolism. Reduction of the aromatic ring of benzoyl-coenzyme A is catalyzed by benzoyl-coenzyme A reductase. This Birch-like reduction is driven by the hydrolysis of 2 ATP molecules. (4) A completely different, still little characterized benzoyl-coenzyme A reductase operates in strict anaerobes, which cannot afford the costly ATP-dependent ring reduction. Key words: aromatic metabolism; benzoyl-CoA pathway; anaerobes; toluene; phenol; benzoate; oxygenases; benzoyl-CoA reductase Introduction ring. Most importantly, the aromatic ring is cleaved by dioxygenases. Aromatic compounds comprise a wide variety of Under microaerobic conditions, facultative aer- natural and synthetic compounds that can serve as obes use another so-called hybrid type of aero- substrates for bacterial growth (FIG. 1). The interest in bic metabolism of benzoate, phenylacetate, and an- cellulose degradation has fostered scientific interest in thranilate (2-aminobenzoate). This metabolism does the degradation of the aromatic copolymer lignin in not require oxygen for ring cleavage. All interme- lignocellulose. Whereas lignin is metabolized mainly diates of these pathways are coenzyme A (CoA) by fungi, bacterial metabolism is thought to be limited thioesters. Dearomatization is catalyzed by an oxyge- to the metabolism of low-molecular-weight aromatic nase/reductase acting on benzoyl-CoA, phenylacetyl- compounds. The diversity of aromatic metabolism is CoA, and 2-aminobenzoyl-CoA, respectively, followed larger than previously thought. So far, besides the well- by an oxygen-independent ring cleavage. studied aerobic type, four types of aromatic metabolism In anoxic water, groundwater, sediments and parts are known. The distribution of these pathways depends of soil, aromatic compounds are metabolized by fac- on the availability of oxygen, on the availability of al- ultative aerobes and phototrophs in the absence of ternative electron acceptors for anaerobic respiration, oxygen in a purely reductive rather than oxidative and also on the rapid fluctuation of oxic and anoxic process. Essential to anaerobic aromatic metabolism conditions. is the replacement of all oxygen-dependent steps by The well-studied aerobic aromatic metabolism is an alternative set of reactions and the formation of characterized by the extensive use of molecular oxy- different central intermediates. These pathways in- gen as cosubstrate for oxygenases, which introduce hy- volve a series of unprecedented enzymes. Notably,two- droxyl groups to facilitate the oxidative cleavage of the electron reduction of the aromatic ring of benzoyl-CoA is driven by the hydrolysis of two molecules of adeno- sine triphospate (ATP). The cyclic, nonaromatic prod- Address for correspondence: Georg Fuchs, Microbiology, Fac- uct formed becomes hydrolytically opened and finally ulty of Biology, University of Freiburg, Schaenzelstr. 1, D-79104 is oxidized to three molecules of acetyl-CoA. + + Freiburg, Germany. Phone: 49 761 203 2649; fax: 49 761 203 2626. Another reductive metabolism is found in strict [email protected] Dedicated to Lars Ljungdahl. anaerobes. Here again, benzoyl-CoA is a central Ann. N.Y. Acad. Sci. 1125: 82–99 (2008). C 2008 New York Academy of Sciences. doi: 10.1196/annals.1419.010 82 Fuchs: Metabolism of Aromatic Compounds 83 FIGURE 1. Sources of aromatic compounds in nature and man-made aromatic compounds derived from fossil material. intermediate of aromatic metabolism. However, when zymes, transcriptional control, genetic organization, one considers growth of strict anaerobes on benzoate, distribution of the pathways, and ecological, evolution- an energetic problem becomes evident: Facultative aer- ary, and applied aspects. obes, such as denitrifyers and phototrophs, spend four ATP equivalents to activate benzoate as CoA thioester (two ATP equivalents) and to reductively dearomatize Aerobic Aromatic Metabolism the ring (another two ATP equivalents). Their energy metabolism, anaerobic respiration, such as nitrate res- The use of molecular oxygen in the cleavage of the piration, yields many more than four ATP equivalents aromatic ring and in hydroxylation reactions in general per one benzoate metabolized; phototrophs conserve is widely distributed in nature, notably when inert or energy by photophosphorylation. In contrast, strict recalcitrant compounds and chemical bonds need to be anaerobes gain fewer than four ATP equivalents out of attacked. Substrates, whose metabolism normally re- one benzoate, which is metabolized via three molecules quires molecular oxygen, include aromatic, hydrocar- of acetyl-CoA plus one CO2. Yet, they still require two bon, and ether compounds. Oxygenases and oxygen ATP equivalents for benzoyl-CoA formation. How- as cosubstrate in hydroxylation reactions have been ever, they cannot spend another two ATP equivalents known since the seminal work of Hayaishi (FIG.2).1 for the reductive dearomatization of benzoyl-CoA, be- In the case of aromatic metabolism, the large va- cause otherwise their energy metabolism would be riety of substrates is channeled via peripheral (up- energy-consuming rather than energy-providing. The per) pathways into a few central intermediates. These outlines of this postulated new principle of benzoyl- peripheral pathways make extensive use of oxygen, CoA reduction are just emerging. which is required by monooxygenases and dioxyge- Most of the novel enzymatic reactions have counter- nases/reductases. The aromatic ring of the central parts in organic chemistry (“Chemistry-inspired Biol- intermediates contains two phenolic hydroxyl groups ogy”). The chemical principles are modified according next to each other, or one hydroxyl group next to a car- to biological constraints, for example, the limits of the boxyl. Examples are catechol (1,2-dihydroxybenzene), redox potential of the cellular electron carriers, the use protocatechuate (3,4-dihydroxybenzoic acid), and gen- of water as solvent at relatively moderate temperature, tisate (2,5-dihydroxybenzoic acid). These free interme- or the low ambient substrate concentrations. diates are substrates of ring cleaving dioxygenases of This short review gives an overview of the different the central (lower) pathways. Ring cleavage may occur strategies, with a focus on the anaerobic pathways. It between the two hydroxyl groups (ortho cleavage) or does not cover the classic aerobic pathways and other next to one of the hydroxyl groups (meta clavage). This important aspects, such as transport, regulation of en- metabolism was established decades ago.2–6 84 Annals of the New York Academy of Sciences FIGURE 2. Aerobic monooxygenase and dioxygenase reactions in aromatic metabolism. Introduction of phenolic hydroxyl groups and ring cleavage both depend on molecular oxygen. Aromatic Metabolism Under nonaromatic intermediate. The subsequent ring cleav- Microaerobic Conditions: The Hybrid age reaction has not been studied yet. Pathways These CoA thioester–dependent pathways may be advantageous under fluctuating oxic/anoxic condi- A few substrates have been recognized to be metab- tions. The pathways allow flexibility and rapid adap- olized by facultative aerobes in a different way. These tation to fluctuating oxygen levels, since both oxic and so-called hybrid pathways still make use of oxygen to anoxic situations require substrate CoA thioesters. If introduce hydroxyl groups, as in the classic aerobic the classic pathway of, for example, benzoate would pathways. At the same time, the aromatic ring is re- operate, the shift from anoxic to oxic conditions would duced and CoA thioesters are used, as in the anaero- result in the accumulation of benzoyl-CoA, since bic metabolism (see later in the chapter). Ring cleavage the ring-reducing enzyme benzoyl-CoA reductase of also does not require oxygen. The new hybrid pathway the anoxic pathway is oxygen-sensitive and therefore of benzoate is shown in FIGURE 3. would be inactive (see later in the chapter). All cellular Denitrifying facultative aerobes (and possibly oth- CoA would be trapped in this dead-end product, since ers) convert phenylacetate,7–14 benzoate,15–18 and benzoate-CoA ligase is oxygen-insensitive and would 2-aminobenzoate (anthranilate)19–24 to their CoA still operate under oxic conditions. CoA depletion ul- thioesters first. Bacteria may even exclusively me- timately would be lethal. In addition, benzoyl-CoA tabolize phenylacetate via this new principle; the oxygenase/reductase, the enzyme that dearomatizes more conventional pathway—via ring hydroxylation to benzoyl-CoA, has a high affinity for oxygen, and there- homogentisate

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