Characterization of the Maleylacetate Reductase MacA of Rhodococcus opacus 1CP and Evidence for the Presence of an Isofunctional Enzyme

Maleylacetate reductases (EC 1.3.1.32) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene macA, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP. Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcus maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of macA, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the R. opacus maleylacetate reductase gene macA clearly is not part of a specialized chlorocatechol gene cluster.Maleylacetate reductases (EC 1.3.1.32) have long been known to be involved in the degradation of chloroaromatic compounds via chlorocatechols as intermediates (10, 31). By reduction of a carbon-carbon double bond they form 3-oxoadipate, a metabolite also of catechol catabolism, and thus compensate for the different oxidation states of chlorinated and nonchlorinated compounds. 2-Chloromaleylacetate, which is formed during turnover of several dichlorocatechols, is initially reductively dechlorinated and then reduced to 3-oxoadipate in a second reaction (22, 47).Corresponding to the biochemical function in chlorocatechol degradation, the following maleylacetate reductase genes have been shown to be associated with dioxygenase, cycloisomerase, and dienelactone hydrolase genes as components of specialized chlorocatechol catabolic operons: tfdF and tfdF_II on pJP4 from the 2,4-dichlorophenoxyacetate-utilizing strain Ralstonia eutropha (Alcaligenes eutrophus) JMP134 (29, 33, 37, 44), tcbF on pP51 from the 1,2,4-trichlorobenzene-degrading strain Pseudomonas sp. strain P51 (45), and clcE from the 3-chlorobenzoate catabolizing strains Pseudomonas sp. strain B13 and Pseudomonas putida AC866(pAC27) (15, 20, 21). Catechol degradation, in contrast, does not require a maleylacetate reductase activity, and corresponding genes do not belong to the known catechol operons. Thus, while at least two of the chlorocatechol catabolic enzymes, i.e., the dioxygenases and cycloisomerases, appear to have been recruited from catechol catabolism, maleylacetate reductase genes must have had a different origin and original function (34).The postulated original function of the maleylacetate reductases is still under discussion. In bacteria, these enzymes have been shown to play a role, for example, in quinol, resorcinol, and 2,4-dihydroxybenzoate degradation (6, 25, 41). Other aromatic growth substrates involving the action of maleylacetate reductase are more exotic, since they carry a fluorine substituent (35), a sulfo group (14), a nitro group (18, 40), or several chlorine substituents (8, 26, 48). Maleylacetate reductase genes have been shown to be part of a specialized gene cluster for 2,4,5-trichlorophenoxyacetate degradation (8, 9) and of a gene cluster for hydroxyquinol conversion which contributes to 4-nitrophenol turnover (4).The chlorocatechol pathway of the chlorophenol-utilizing strain Rhodococcus opacus (erythropolis) 1CP obviously evolved functionally convergent to the corresponding pathway in the proteobacteria mentioned above (13, 39). Thus, it is not surprising that the chlorocatechol gene cluster of strain 1CP is organized differently from the corresponding proteobacterial operons; in fact, its characterization showed that it does not comprise a maleylacetate reductase gene (13). Thus, the nature of the gene cluster(s) encoding a maleylacetate reductase in R. opacus remained to be elucidated. Such gene clusters could complement otherwise incomplete pathways, and they might also have provided the source from which the maleylacetate reductase gene was recruited during evolution of dedicated pathways, such as the proteobacterial chlorocatechol catabolic route.(Some of the results presented here have previously been reported in a preliminary communication [38].)