Research for the molecular evolution of the adaptive mutants obtained after continuous culture of cumene degrader on biphenyl.

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Cumene ( isopropylbenzene ) is an aromatic hydrocarbon with a chemical structure close to those of toluene and biphenyl. P.fluorescens IP01 can grow on cumene or toluene as a sole carbon and energy source but not on biphenyl. We surmised that the growth substrate specificity of strain IP01 is ascribed to the substrate specificity of the meta-cleavage compound hydrolase (CumD ).In this study, we attempted to determine the residue responsible for the subatrate specificity in CumD by means of site-directed mutagenesis. Amino acid sequence comparison between CumD and other hydrolases led us to identify several conserved residues likely to have a functional role in the catalytic center of CumD,that is, three amino acids, Ser^<103>, Asp^<224>, His^<252>, were found to be arranged in a sequential order similar to that in serine hydrolases. Substitutions of each of these three residues by Ala decreased enzymatic activity on hydrolisis of meta-cleavage compound of cumene (HOMODA ) below 5%. The … More se results suport the hypothesis that Ser^<103>, Asp^<224>, His^<252> constitute the catalytic triad of CumD.Substitution of Ile^<256> by Trp and that of Trp^<143> by Phe increased enzymatic activity on hydrolysis of meta-cleavage compound of biphenly ( HOPDA ). This result suggests that Ile^<256> and Trp^<143> are the key residues to alter the substrate specificity of CumD so as to hydolase HOPDA.It was also indicated that substitution of Val^<227> by Ile increased enzymatic activity on hydrolysis of HOMODA.This result suggests that Val^<227> is Iocated nearby the catalytic triad topologically, and is involved in the substrate-binding site in CumD.We obtained 25 mutants degrading efficiently biphenyl after continuous culture of IP01 on biphenyl containing a small amount of cumene. In these mutants, it was predicted that substrate specificity of CumD had been altered as a result of mutation for the cumD gene, but any mutation in cumD was not detected in sequence analysis. Comparison of growth of 5 mutants ( selected from 25 mutants ) with that of wild-type IP10 indicates that the activity of a series of enzymes in the cumene degradation pathway, or mechanism of transcriptional control of the cum gene, may have altered in the 5 mutants. Less