Characterization of the maleylacetate reductase MacA of Rhodococcus opacus 1CP and evidence for the presence of an isofunctional enzyme

Abstract

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.

FIG. 1

Restriction and genetic map of the inserts of the original plasmids pMARRE1 and pMARRE3 carrying R. opacus 1CP DNA in the multiple cloning site of pBluescript II SK(+) (vector segments are indicated by shading). The insert of subclone pMARRE5 is shown, as is the PCR product (black) cloned to give pMARRE0, which was used as a digoxigenin-labeled probe for cloning. The positions and orientations of open reading frames are indicated by arrows below the restriction maps. The insert of pMARRE2 (not shown) extends from the XhoI site of pMARRE1 ca. 7 kb to the right.

FIG. 2

Multiple sequence alignment of various maleylacetate reductases as calculated by the CLUSTAL program. Amino acids identical in all sequences are highlighted. Numbers above the sequences refer to positions in the alignment, not in a single sequence. Asterisks indicate one pattern typical for type III (iron-containing) alcohol dehydrogenases, [AP]-x(6)-[STAP]-x(5,6)-P-x(4)-[AIV]-x-[GST]-x(2)-D-[AI]-[LIVM]-x(4)-E (Prosite entry PDOC00059; last update, November 1995). A second pattern (Δ) typical for this enzyme family could be important for binding ferrous iron: H-x(2)-[SAE]-[HY]-x(2)-[SGA](2)-x(5)-H-G. Interesting differences from these patterns are marked by exclamation marks. Accession numbers and references for the published sequences (order as in the alignment): AF019038 (21), M57629 (45), M35097 (29), U19883 (8), U16782 (44). MAR, maleylacetate reductase.