COMPARISON OF THE NUCLEOTIDE-SEQUENCES OF THE META-CLEAVAGE PATHWAY GENES OF TOL PLASMID PWW0 FROM PSEUDOMONAS-PUTIDA WITH OTHER META-CLEAVAGE GENES SUGGESTS THAT BOTH SINGLE AND MULTIPLE NUCLEOTIDE SUBSTITUTIONS CONTRIBUTE TO ENZYME EVOLUTION

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzyme s required for the oxidation of toluene and xylenes. The structural ge nes for these catabolic enzymes are clustered into two operons, the xy lCM ABN operon, which encodes a set of enzymes required for the transf ormation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJ QKIH operon, which encodes a set of enzymes required for the transform ation of benzoate/toluates to Krebs cycle intermediates. The latter op eron can be divided physically and functionally into two parts, the xy lXYZL cluster, which is involved in the transformation of benzoate/tol uates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is inv olved in the transformation of (methyl)catechols to Krebs cycle interm ediates. Genes isofunctional to xylXYZL are present in Acinetobacter c alcoaceticus, and constitute a benzoate-degradative pathway, while xyl TEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pa thway and a naphthalene-degradative pathway are present on plasmid pVI 150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, res pectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQ KIH genes with other isofunctional genes suggested that the xylTEGFJQK IH genes on the TOL plasmid diverged from these homologues 20 to 50 mi llion years ago, while the xylXYZL genes diverged from the A. calcoace ticus homologues 100 to 200 million years ago. In codons where amino a cids are not conserved, the substitution rate in the third base was hi gher than that in synonymous codons. This result was interpreted as in dicating that both single and multiple nucleotide substitutions contri buted to the amino acid-substituting mutations, and hence to enzyme ev olution. This observation seems to be general because mammalian globin genes exhibit the same tendency.