Neilson, Alasdair H. "Pathways of Biodegradation and Biotransformation" Organic Chemicals : An Environmental Perspective Boca Raton: CRC Press LLC,2000 ©2000 CRC Press LLC 6 Pathways of Biodegradation and Biotransformation SYNOPSIS An attempt is made to describe the pathways used by microor- ganisms to degrade or transform xenobiotics. Most of the major structural groups are considered, including aliphatic, alicyclic, aromatic, and heterocy- clic compounds, including those with oxygen, sulfur, nitrogen, phosphorus, or halogen substituents. Although organochlorine compounds have received most attention, an attempt has been made to include also organobromine compounds; the degradation of organofluoro compounds is discussed sepa- rately since these compounds differ significantly even in their chemical prop- erties from those of the other halogens. Reactions carried out by both aerobic and anaerobic bacteria and—to a somewhat lesser extent by yeasts and fungi—are considered, although no details of the enzyme systems are given. Attention is directed to the pathways that are used by different organisms for the degradation of a given xenobiotic. Investigations using aerobic bacteria have almost invariably been exemplified from the results of experiments using pure cultures, whereas for anaerobic bacteria this has been supple- mented by results using mixed cultures or stable consortia. Some examples are given of the application of biotransformation reactions in biotechnology and of the environmental significance of the biotransformations of xenobiot- ics which may result in metabolites more toxic than their precursors. Finally, an attempt has been made to classify the reactions involved in the degrada- tion of xenobiotics on the basis of well-established chemical transformations, and specific reference has been made to the appropriate sections in which these reactions are discussed in greater detail. Introduction It is desirable to explain both the motivation and the objectives of this chapter since some of the material has already been presented from a different per- spective in preceding chapters: Chapter 4 attempted to provide a general background with a microbiological emphasis, while Chapter 5 filled this out with an outline of procedures for carrying out the appropriate experiments. ©2000 CRC Press LLC This chapter attempts a survey of the pathways by which a range of structur- ally diverse xenobiotics are degraded or transformed by microorganisms; the emphasis is on reactions mediated by bacteria which are the most effective agents in carrying out biodegradation in most natural aquatic ecosystems. It is appropriate to begin by underscoring the two rather different—and possibly conflicting—approaches to addressing problems of biodegradation and biotransformation, and to which attention has already been briefly drawn. These concern the level at which assessments of biodegradability are carried out. On the one hand, conventional tests for assessing ready biodegradability do not provide an adequate base for determining what occurs after release of the compound into natural ecosystems, even though they may be adequate for assessing biodegradability in the municipal treatment systems from which the inocula were taken. Indeed, the effort directed to developing stan- dardized test systems may even have been counterproductive to environ- mental relevance for reasons that have been outlined in more detail in Chapter 5, Section 5.2.1. On the other hand, the comprehensive investigations which have been pur- sued on the physiology, biochemistry, genetics, and regulation of biodegra- dation cannot realistically be incorporated even into an advanced hazard assessment except in a very few instances. An additional problem arises from the immense structural range of organic compounds that are used industrially or have been incorporated into commer- cial products. The skill of the organic chemist is seemingly unlimited and with the inevitable need for new compounds, which attempt to avoid the undesir- able consequences of their traditional counterparts, the number of com- pounds—as well as their structural diversity—seems unlikely to diminish. There is an enormous literature on the microbial degradation and transfor- mation of organic compounds, and it would be attractive to take advantage of this to construct generalizations on the pathways used for the degradation of broad structural classes of xenobiotics. That is the objective of this chapter. This approach has been illustrated by Alexander (1981), and this chapter attempts to provide details that were not possible within the space of that seminal review. In addition, this procedure would have a predictive capabil- ity that is not restricted to compounds which have already been investigated. Although structure–activity relationships are useful for classifying existing data, they have an inevitably restricted potential for application to com- pletely novel chemical structures. Support for this mechanistic approach is provided by its success in assessing the biodegradability of 50 structurally diverse xenobiotics (Boethling et al. 1989). This chapter does not attempt to encompass the enzymology of the reac- tions involved in the degradation of xenobiotics, so that the word pathways is more appropriate than mechanisms: however desirable, discussions of enzy- mology lie both beyond the scope of the present work and the competence of the author. A few parenthetical comments on the enzymology of the reactions have, however, been made if they elucidate the scope and the generality of ©2000 CRC Press LLC the reactions under consideration. Attention has been drawn to the role of free radicals in enzymatic reactions (Section 4.4.4), and there is increasing appreciation of their wider significance in reactions catalyzed by enzymes (Stubbe and van der Donk 1998); examples are given in Sections 6.7.1 and 6.7.3.1. This recognition parallels the development by D.H.R. Barton of radi- cal-mediated reactions in synthetic organic chemistry in which mild condi- tions, high yield, and specificity are combined. Some important details of the reaction pathways involved in degradation have been deliberately omitted in the figures used to illustrate the various sequences: for example, (1) even when the degradation of carboxylic acids takes place through initial formation of the coenzyme-A esters, sequences have depicted the free carboxylic acids and (2) in some cases, although the structures of intermediates have not been rigorously determined, these have been included to illustrate more clearly the structural relationships between the initial substrate and the various metabolites. The presentation is made on the basis of the chemical structure of xenobiotics and is dominated by examples of reactions carried out by aerobic and anaerobic bacteria and—to a lesser extent—aerobic fungi and yeasts; some examples of biotransformation reactions carried out by other microorganisms are given in Chapter 4, Section 4.3, and by higher organisms in Section 7.5. Although anaerobic fungi are known and are certainly important in rumen metabolism (Mountfort 1987), their existence in other habitats does not appear to have been estab- lished and their potential for degrading xenobiotics does not seem to have been explored. Since the emphasis is on degradative pathways, less attention will be devoted to the taxonomic delineation of the var- ious organisms except where these belong to less common taxa while reference has already been made in Chapter 5, Section 5.9 to the serious problems that have been encountered in attempting to classify bacteria of established degradative importance. In addition, no attempt has been made to provide the currently acceptable taxonomic assignment of the organisms that are involved, and the designations used by the authors have been retained with only a few exceptions. Except for the simplest reaction sequences, structural representations of the various pathways are given in the form of flow diagrams rather than by using conventional chemical nomenclature. It is hoped that the reactions are thereby more clearly perceived in geometric terms, particularly to those who are not organic chemists and who are understandably repelled by the seemingly barbarous complexity and apparent incom- prehensibility of systematic organic chemical nomenclature. In the following sections, an account will be presented of the pathways by which xenobiotics are degraded by microorganisms. At the same time, it is essential to bear in mind certain fundamental aspects of the microbiology and biochemistry of the cells carrying out these reactions and, in particular, the role of metabolites that are required for biosynthesis on which continued growth and replication of the organisms ultimately depend. ©2000 CRC Press LLC 1. If an organic compound is to support growth and replication of an organism, it must also provide the necessary metabolic energy and serve as the source of carbon (and, in some cases, also nitrogen or sulfur or phosphorus) for the synthesis of cell material. Details of these metabolic reactions are not given here, and a good account may be found, for example, in Mandelstam, Macquillan, and Dawes (1982). These reactions then determine the extent to which the constituent atoms of xenobiotics are incorporated into the glo- bal carbon, nitrogen, sulfur, or phosphorus cycles; these are not discussed here, and reference may be made to the valuable account of carbon cycles into which the products from the degradation of xenobiotics are incorporated (Hagedorn et al. 1988). Whereas the functional operation of these reactions is a prerequisite for biodeg- radation, biotransformation may be accomplished by nongrowing cells, or in cells growing at the expense of more readily degradable substrates; this has been discussed in Section 4.5.2. 2. Just as there is no single pathway universally used for the catabo- lism of simple substrates such as glucose, there are no unique pathways for the degradation of a given xenobiotic. The following examples may be used to illustrate the considerable differences in the pathways used for the degradation of xenobiotics by bacteria and by fungi, or even by different taxa of bacteria. • The degradation of DDT by Phanerochaete chrysosporium (Bum- pus and Aust 1987) and by Aerobacter aerogenes (Wedemeyer 1967); • The degradation of 2,4-dichlorophenol by Ph. chrysosporium (Val- li and Gold 1991) and by a strain of Acinetobacter sp. (Beadle and Smith 1982); • The degradation of quinoline by pseudomonads and by Rhodo- coccus sp. (Schwarz et al. 1989); • The degradation of tryptophan by Pseudomonas fluorescens that takes place via the β -ketoadipate pathway and by P. acidovorans that utilizes the quinoline pathway (Stanier 1968); • The plethora of pathways for the aerobic degradation of toluene (Section 6.2.1). 3. There is no absolute distinction between the degradative pathways used by aerobic and by anaerobic bacteria. Simple reductions are carried out by organisms with a strictly aerobic metabolism. These include, for example, reductive dechlorination of phenolic com- pounds by R. chlorophenolicus (Apajalahti and Salkinoja-Salonen 1987a), reduction of hydroxylated pyrimidines by P. stutzeri (Xu and West 1992), and degradation of anthranilate by a strain of Pseudomonas sp. that is able to use this as a source of both carbon and nitrogen and degrades the substrate by initial reactions ©2000 CRC Press LLC involving the reduction of the aromatic ring (Altenschmidt and Fuchs 1992b). This should not, however, be interpreted to imply that the underlying cellular metabolism of aerobic and anaerobic microorganisms is necessarily comparable. 4. Although a synopsis of the reactions used by microorganisms for the degradation and transformation of organic compounds is given in Section 6.12, it may be valuable to provide some general comments at this stage. The basic reactions known in organic chemistry provide a suitable background for rationalizing most biochemical reactions—addition, elimination, substitution, oxida- tion, reduction, and rearrangement—and all of these can be medi- ated by microorganisms although, for example, degradation involving addition reactions is rather unusual. The degradation of aliphatic (and alicyclic) and aromatic (including heterocyclic) com- pounds has been treated separately in this chapter, since both their chemistry and their microbial degradation pathways differ signif- icantly. The following categorical summary may illustrate the broad types of reactions that are most commonly encountered and may serve as a prelude to the more–detailed discussions of indi- vidual groups of compounds that follow. A detailed summary is given in Section 6.12. Oxygenation —Most organic xenobiotics are relatively highly re- duced compounds so that their degradation to CO 2 and H 2 O inevitably involves introduction of oxygen into the molecule either by monooxygenation or dioxygenation from O 2 or by hydroxylation from H 2 O. Whereas oxygenation is clearly re- stricted to aerobic conditions, hydroxylation can be accom- plished both aerobically and anaerobically. It should be appre- ciated that oxidation can occur under anaerobic conditions provided that the redox balance is preserved within the system. Methanogenesis is the terminal—although complex—step in the reduction of the precursors (CO 2 or acetate) that are produced by the degradation of more complex substrates. Dehydrogenation or desaturation —Under aerobic conditions, dehy- drogenations may be involved and these may be important un- der anaerobic conditions. Dehalogenation —The degradation of compounds carrying haloge- nated substituents will involve loss of halogen that may occur by elimination or by displacement reactions; these may be re- ductive, oxidative, or hydrolytic. Rearrangement —These are particularly important among anaerobic bacteria where they involve coenzyme-B 12 . The unrelated rear- rangement of the substituents on aromatic rings (the NIH shift) is well established particularly among fungi. ©2000 CRC Press LLC A cardinal issue for the successful biodegradation of xenobiotics is the bioenergetics of these reactions, although this aspect is not discussed here. Whereas the synthesis of ATP under aerobic conditions is at least formally straightforward, a much greater range of mechanisms operates under anaer- obic conditions where ATP may be generated, for example, from intermedi- ate acyl phosphates (generally acetyl phosphate), carbamyl phosphate, or 10-formyl tetrahydrofolate (Section 4.2.1). As has already been emphasized, citations to the literature are eclectic rather than complete. Comprehensive reviews of many of the groups of com- pounds have been provided in the books and in the review articles that are given at the beginning of the reference list in Chapter 4, and these should be consulted for further details. 6.1 Aerobic Degradation of Nonaromatic Hydrocarbons 6.1.1 Alkanes There is an enormous literature on the microbial degradation of alkanes; this has been motivated by aims as diverse as the utilization of microorganisms for the production of single-cell protein or their application to combating oil spills. Both the number and the taxonomic range of microorganisms are equally impressive, and they include many different taxa of bacteria, yeasts, and fungi. Extensive reviews that cover most aspects have been presented (Ratledge 1978; Britton 1984). The simplest alkane is, of course, methane, but the pathways for its deg- radation and assimilation do not reflect this structural simplicity. In outline, the pathway of degradation is straightforward and involves successive oxi- dation to methanol catalyzed by methane monooxygenase, and successive dehydrogenation to formaldehyde and formate. The cells must, however, be capable of synthesizing cell material from the substrate so that some fraction of the C 1 metabolites must also be assimilated. Several distinct pathways for this have been described, but these are merely summarized here since a comprehensive and elegant presentation of the details has been given (Anthony 1982): 1. The ribulose bisphosphate pathway for the assimilation of CO 2 which is identical to the Benson–Calvin cycle used by photosyn- thetic organisms; 2. The ribulose monophosphate cycle for the incorporation of form- aldehyde; 3. The serine pathway for the assimilation of formaldehyde. ©2000 CRC Press LLC Methane monooxygenase may exist in either soluble (sMMO) or particu- late (pMMO) forms. These display different substrate ranges and different rates of transformation, and most methanotrophs express only the latter form of the enzyme (Hanson and Hanson 1996). The role of Cu in determining whole-cell activity of pMMO is discussed in Chapter 5, Section 5.2.4. The sol- uble methane monooxygenase from both type I and type II methanotrophs is a three-component system comprising a nonheme iron hydroxylase contain- ing a bridged oxo-bridged binuclear Fe cluster (A), a metal-free protein com- ponent without redox cofactors (B), and a NADH reductase (C), containing FAD and a [2Fe–2S] cluster (Fox et al. 1989). One additional aspect is the wide spectrum of substrates which can be metabolized by the methane monooxygenase system, and some illustrative examples are given in Figure 6.1. Attention has already been drawn in Chap- ter 4, Section 4.3.2 to the similarity of this enzyme to that involved in the oxi- dation of ammonia, while the broad substrate specificity of cyclohexane oxygenase is noted again in Section 6.1.2. The initial hydroxylation of alkanes is mediated by both membrane-bound and soluble hydroxylases, and the genetics of alkane hydroxylation and alkanol dehydrogenation in pseudomonads is complex involving in Pseudomonas oleovorans the loci alkA, alkB, alkC, alkD, alkE (Fennewald et al. FIGURE 6.1 Examples of the reactions catalyzed by methane monooxygenase. ©2000 CRC Press LLC 1979; Kok et al. 1989). In P. putida that carries the OCT plasmid, there is dupli- cation of some of the loci: those for alkane hydroxylation (alkA, alkB, alkC) and for alkanol dehydrogenation (alcO) occur on the plasmid, whereas those for alcA and alcB, and for aldehyde dehydrogenation (aldA, aldB) occur in the chromosome (Grund et al. 1975). (Note the different symbols used for genetic loci in these studies.) The corresponding genes on the OCT plasmid of P. oleovorans and in Acinetobacter sp. strain ADP1 have been discussed in Chapter 4, Section 4.4.1.1. There is also some structural similarity between the nucleotide sequence for alkane hydroxylase and the subunits of the monoox- ygenase coded by xylA and xylM that are involved in the side-chain oxidation of toluene and xylene by P. putida PaW1 (Suzuki et al. 1991). The conversion of methanol to formaldehyde is carried out by methanol dehydrogenase. A complex array of genes is involved in this oxidation, and the dehydrogenase contains pyrroloquinoline quinone (PQQ) as a cofactor (refer- ences in Ramamoorthi and Lidstrom 1995). The details of its function must, however, differ from methylamine dehydrogenase that also contains a quino- protein–tryptophan tryptophylquinone (TTQ) (Section 4.4.4). The initial reac- tions involved in the metabolism of higher alkanes (> C 1 ) are formally similar to those used for the metabolism of methane, and the soluble alkanol dehydro- genases also contain PQQ (references in Anthony 1992). Enzymatically, how- ever, the details may be more complex since, for example, a number of distinct alcohol and fatty acid dehydrogenases have been isolated from an Acineto- bacter sp. during the metabolism of hexadecane (Singer and Finnerty 1985a,b). Further degradation of the carboxylic acid following oxidation of the alde- hydes involves a β -oxidation with successive loss of acetate residues (Figure 6.2). A structurally wide range of hydrocarbons may be degraded by micro- organisms including linear alkanes with both even numbers of carbon atoms up to at least C 30 , some odd numbered alkanes including the plant wax C 29 H 60 (Hankin and Kolattukudy 1968), and branched alkanes such as pristane (2,6,10,12-tetramethylpentadecane) (McKenna and Kallio 1971; Pirnik et al. 1974). A number of details merit brief comment. 1. Yeasts are able to degrade long-chain alkanes. The initial hydrox- ylation is carried out in micrososmes by cytochrome P-45O, while degradation of the alkanoate is carried out in peroxisomes that contain the β -oxidation enzymes: alkanoate oxidase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. Further details are given in Chapter 4, Sections 4.4.1.2 and 4.4.4. FIGURE 6.2 Outline of the metabolism of alkanes. ©2000 CRC Press LLC 2. In some cases, reaction between the initially formed alkanol and its oxidation product, the alkanoic acid, may produce esters which are resistant to further degradation (Kolattukudy and Hankin 1968). 3. For complete degradation and assimilation of the products into anabolic pathways, the cells must clearly be capable of synthesizing the appropriate enzymes. When β -oxidation results in the produc- tion of acetate, cells must be capable of synthesizing the enzymes of the glyoxylate cycle. When odd-membered alkanes are oxidized, propionate is also produced and its further degradation may follow a number of alternative pathways (Figure 6.3) (Wegener et al. 1968). A further alternative has been demonstrated in Escherichia coli K12 in which the initial reaction is condensation of propionyl-CoA with oxalacetate to form methylcitrate that is then converted into succi- nate and pyruvate (Textor et al. 1997). The enzyme was identified as citrate synthetase II which has an established role in the metab- olism of propionate by the yeast Candida lipolytica (Uchiyama and Tabuchi 1976). 4. Oxidation of compounds such as pristane proceeds by both β -oxi- dation and ω -oxidation (McKenna and Kallio 1971; Pirnik et al. 1974) (Figure 6.4). Pristane may also be degraded under nitrate- reducing conditions in microcosms and enrichment cultures (Breg- nard et al. 1997). 5. The existence of chain branching may present an obstacle to deg- radation, although this can be circumvented by a carboxylation pathway (Figure 6.5) (Fall et al. 1979) that is formally comparable to that illustrated above for the degradation of propionate. Carbox- ylation is also used in one of the pathways used by Marinobacter FIGURE 6.3 Pathways for the biodegradation of propionate. [...]... (Bachofer and Lingens 1975), although details of the enzyme are not fully resolved (Fukumori and Saint 1997) The range of substituted anilines that have been examined includes the following: 2-, 3-, and 4-chloroanilines and 4- uoroaniline by a Moraxella sp strain G (Zeyer et al 1985), 3-, and 4-methylanilines by P putida mt-2 (McClure and Venables 19 86) , 2-methylaniline and 4-chloro-2methylaniline by... mammalian systems (Smith and Rosazza 1983), there is one very significant difference—and that is the stereochemistry of the products trans-1,2Dihydroxy-1,2-dihydroanthracene and trans-1,2-dihydroxy-1,2dihydrophenanthrene are formed from the hydrocarbons by FIGURE 6. 33 Biotransformation of 7-methylbenz[a]anthracene by Cunninghamellaelegans ©2000 CRC Press LLC FIGURE 6. 34 Alternative pathways for the biotransformation... transformation of caryophyllene oxide by Botrytis cinerea Although most of the reactions were hydroxylations or epoxidations, two involved transannular reactions (1) between the C-4-epoxide oxygen and C-7 and (2) between the C-4-epoxide and C-13 with formation of a caryolane (Figure 6. 20) (Duran et al 1999) FIGURE 6. 18 Degradation of α-pinene ©2000 CRC Press LLC FIGURE 6. 19 Degradation of tropine 6. 1.3... α -pinene, ©2000 CRC Press LLC FIGURE 6. 14 Hydroxylation of (a) 5,5-difluorocamphor, (b) adamantane, and (c) patchoulol FIGURE 6. 15 Hydroxylation of 5-bromo- 7- uoronorbornanone FIGURE 6. 16 Hydroxylation of azabrendane derivative ©2000 CRC Press LLC FIGURE 6. 17 Biodegradation of a C3-oxygenated bile acid although some strains of Pseudomonas sp degrade this by rearrangement to limonene, oxidation, and... benzoyl formate and benzoate that is further metabolized via catechol and the β-ketoadipate pathway (Figure 6. 32a) (Hegeman 1 966 ) Both enantiomers of mandelate were degraded through the activity of a mandelate racemase (Hegeman 1 966 ), and the racemase (mdlA) is encoded in an operon that includes the following two enzymes in the pathway of degradation, S-mandelate dehydrogenase (mdlB) and benzoylformate... (1-chloro-2,3-epoxypropane) by the same strain (Small et al 1995) 5 Rhodococcus sp strain AD45 carried out the transformation of 2-methyl-1,3-butadiene (isoprene), and both cis- and trans-dichloroethenes to the epoxides (van Hylckama Vlieg et al 1998) The degradation of the dienes takes place by a pathway involving a glutathione S-transferase that is able to react with the epoxides and a conjugate-specific... by rearrangement from the initially produced trans dihydrodiol may be conjugated to form sulfate esters or glucuronides (Cerniglia et al 1982a; Golbeck et al 1983; Cerniglia et al 19 86; Lange et al 1994 ), and the less common glucosides have also been identified: 1-phenanthreneglucopyranoside is produced from phenanthrene by C elegans (Cerniglia et al 1989) and 3-( 8-hydroxyfluoranthene)-glucopyranoside... quaternary carbon atom (Figure 6. 16) (Archelas et al 1988) 5 Penicillium lilacinum transformed testosterone successively to androst-4-ene-3,17-dione and testololactone (Prairie and Talalay 1 963 ): once again, the oxygen atom is introduced into ring D at the quaternary position between C-13 and C-17 The degradation of sterols and related compounds has been extensively studied and, for bile acids, involves... and a conjugate-specific dehydrogenase that produces 2-glutathionyl2-methylbut-3-enoate (van Hylckama Vlieg et al 1999) Epoxides may be formed from alkenes during degradation by Pseudomonas oleovorans, but oct-1,2-epoxide is not further transformed, and degradation of oct-1-ene takes place by ω-oxidation (May and Abbot 1973; Abbott and Hou 1973) The ω-hydroxylase enzyme is able to carry out either hydroxylation... the (original) 6- methyl group The second involves introduction of oxygen between C-2 and C-3 by a Baeyer-Villiger-type oxidation that is noted in Section 6. 1.2 Hydrolysis of the lactone is followed by dehydrogenation of the alkanol and subsequent degradation 6 A number of substituted 2,2-bisphenylpropanes are degraded by oxidation and cleavage at the quaternary carbon atom (Figure 6. 6) (Lobos et al . involved transannular reactions (1) between the C-4-epoxide oxygen and C-7 and (2) between the C-4-epoxide and C-13 with formation of a caryolane (Figure 6. 20) (Duran et al. 1999). FIGURE 6. 17 Biodegradation. odd numbered alkanes including the plant wax C 29 H 60 (Hankin and Kolattukudy 1 968 ), and branched alkanes such as pristane (2 ,6, 10,12-tetramethylpentadecane) (McKenna and Kallio 1971;. FIGURE 6. 14 Hydroxylation of (a) 5,5-difluorocamphor, (b) adamantane, and (c) patchoulol. FIGURE 6. 15 Hydroxylation of 5-bromo- 7- uoronorbornanone. FIGURE 6. 16 Hydroxylation of azabrendane