| Summary: | <I>Rhodococcus rhodochrous </I>N75, a nocardioform actinomycete, metabolises methyl-substituted aromatic compounds via the modified <I>ortho-</I>cleavage pathway whereby a novel 4-methylmuconolactone methyl-isomerase catalyses the conversion of 4-methylmuconolactone to 3-methylmuconolactone in order to circumvent the accumulation of the 'dead-end' metabolite 4-methylmuconolactone which is found in some other species of bacteria. <I>R. rhodochrous </I>N75 was found to transform a range of alkyl-substituted catechols to the corresponding muconolactones since the two enzymes initiating the degradative pathway, catechol 1,2-dioxygenase and <I>cis,cis-</I>muconate cycloisomerase, possess broad substrate specificities. Catechol 1,2-dioxygenase was highly purified from cells of <I>R. rhodochrous </I>N75 grown at the expense of benzoate and <I>p-</I>toluate as the sole sources of carbon. A single catechol 1,2-dioxygenase was found to be induced with either grown substrate. The enzyme has an estimated M<SUB>r</SUB> of 71,000 consisting of two identical subunits. Catechol 1,2-dioxygenase from <I>R. rhodochrous </I>N75 exhibits some unusual properties including: broad substrate specificity, extradiol cleavage activity with 4-methylcatechol and low K<SUB>m</SUB> values for halocatechols, suggesting that this enzyme is distinct from other known catechol and chlorocatechol 1,2-dioxygenases. <I>cis,cis-</I>Muconate cycloisomerase was purified to homogeneity from cells of <I>R. rhodochrous </I>N75 grown at the expense of benzoate and <I>p-</I>toluate as the sole sources of carbon. A single cycloisomerase was also found to be induced in this organisms with no isoforms being detected when <I>R. rhodochrous </I>N75 was grown on either benzoate and <I>p-</I>toluate. The enzyme is hexameric with a single subunit M<SUB>r</SUB> of 40,000. <I>cis,cis-</I>Muconate cycloisomerase from <I>R. rhodochrous </I>N75 displays strict regio- and stereospecificity whereby <I>cis,cis-</I>muconate is cyclised to (3S)-muconolactone and 2-methyl- and 3-methyl-substituted muconates are cyclised to 2-methyl- and 4-methyl-substituted muconolactones by 1,4 - and 3.6-cyclisation respectively.
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