INFLUENCE OF SIDE CHAINS AND MODIFICATION OF SUBSTITUENTS BEFORE RING CLEAVAGE.

The principal reactions involved in the transformation of benzene ring substituents include p-oxidation, oxidative déméthylation, epoxidation of carbon-carbon double bonds and hydroxylation. These metabolic steps are representative of the few mechanisms used to deal with a diverse array of chemicals. The bacterial species involved and the size and type of

substituent governs whether the side chain remains intact, or is transformed or eliminated before ring cleavage.

1.10.1 Methyl, Methoxv and Carboxv Substituents.

Methyl groups may either be oxidised or remain intact during hydroxylation of the aromatic nucleus. Kitagawa (1956) showed that a strain of aeruginosa was able to oxidise toluene, benzyl alcohol, benzaldehyde and benzoate at approximately equal rates (Figure 1.42a). More recently, Worsey and Williams (1975) showed that P. putida (arvilla) mt-2 used a similar route for the catabolism of toluene but that the monooxygenase was encoded on a TOL plasmid. Toluene may also be degraded via

dihydroxylation of the aromatic nucleus, leaving the methyl moiety intact. Claus and Walker (1964) first concluded that this may occur using a Pseudomonas sp. and an Achromobacter sp. from

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soil (Figure 1.42b). Subsequently, the pathway has been verified and the intermediate, toluene cis-qlycol. has been identified. Similarly, larger alkyl side chains may either remain intact during the formation of the corresponding catechol or undergo oxidation prior to catechol production. In the latter case, carboxylic acids are then formed by oxidation of the terminal methyl group. Provided that extensive branching does not exist, the larger carboxyalkyl substituents can undergo 0-oxidation, eventually yielding phenylacetic or benzoic acid. Direct oxidation of the aromatic nucleus of alkyl benzenes yield a substituted cis-qlycol. However, it has been shown that longer chain alkyl benzenes may prevent attack on the aromatic nucleus. Isopropyl benzene and n-butyl benzene have been shown to be oxidised by toluene-grown cells but the relative rates were much slower (Gibson et al.. 1968). Work using microbial cells

isolated on aromatic substrates possessing longer side chains has not significantly affected the premise that longer alkyl side chains are removed prior to ring cleavage. DeFrank and Ribbons (1977) have shown that £-cymene grown P^_ putida strain PL form E-cumate prior to the formation of the dihydrodiol and 2,3-dihydroxy p-cumate, thus the isopropyl group remains intact during cleavage. Similarly, Catelani et al. (1977) showed that the quaternary carbon in tert-butvl benzene remained intact during ring cleavage by Achromobacter A2.

A carboxyl substituent of an aromatic acid may remain unaltered before ring cleavage, forming protocatechuate or it may be eliminated as in the TOL plasmid-encoded degradation of xylene or toluene.

Figure 1-42- Possible pothwovs for the dearodotion of toluene-

Fig- 1-42b Dioxygenase

Fig. 1-42a Monooxygenase

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give the parent phenol, with the concomitant liberation of the alkyl moiety as an aldehyde. Thus, vanillic acid is converted to protocatechuate by the loss of its methoxy group in the form of formaldehyde. Such oxidative demethylation has previously been discussed (Section 1.9.1).

1.10.2 Halogen substituents.

The elimination of halogen substituents from aromatics is of special interest because their presence usually adds to the recalcitrance of hydrocarbons. Many of the haloaromatics in the environment arise by chemical synthesis and, as such, must be viewed as xenobiotic compounds. These xenobiotics can be

metabolized fortuitously with incomplete degradation or they can be totally degraded. This may occur via a sequence of

co-metabolic transformations through participation of more than one organism or by the complete catabolic machinery of a single organism and thus be utilised as sole carbon and energy source.

Many haloaromatics are degraded largely by fortuitous metabolism. The enzymes which normally serve a physiological role can often act on xenobiotic compounds provided structural analogy allows substrate binding and comparable reactivity of functional groups. It is well established that enzymes are not absolutely specific with respect to substrate binding so that substrates with xenobiotic structural elements may be bound. This facilitates the degradation of xenobiotics and allows the manufacture of high value-added halo-organic compounds using a biocatalyst. An example of the broad substrate specificity which may be manipulated in this way is provided by the methane

in Table 1.7 illustrate that halogenated alkanes can be converted but the relative velocities of oxidation of

substituted methane decreases sharply with the nature of the substituent.

The enrichment procedure used largely governs whether a particular isolate is able to co-metabolise a halo-aromatic compound. Thus, toluate-oxidising bacteria exhibit higher co-metabolic activities on chloro-substituted analogues than ordinary benzoate degraders. This is illustrated in Table 1.8 showing that Alcaliqenes eutrophus, induced on benzoate, has a low turnover rate of substituted benzoates. Kinetic analyses have shown that reaction rates are predominantly decreased by the steric effects of the substituents (Reineke and Knackmuss, 1978). In contrast, dioxygenation of substituted benzoic acids by toluate-grown cells of P. putida mt-2 is mostly undisturbed by the steric effects of chlorine as a substituent. Elimination of the halogen substituent is essential for the complete

mineralization of the substrate and can be accomplished in a number of ways. The mechanism of direct dechlorination by microbial monooxygenases is suggeted to follow the NIH-shift. This has been studied with aromatic substrates labelled in specific positions with deuterium. A frequent consequence of hydroxylation is an intramolecular migration or shift of the group displaced by hydroxyl to an adjacent position on the aromatic ring (Daly et al.. 1972). Initial dehalogenation of halo-aromatic compounds have rarely been found and these cases are suggested to follow the pathway outlined in Figure 1.43. This pathway has been suggested to operate in Pseudomonas sp. B13 during adaptation to 2-fluorobenzoate as sole carbon source

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