The physiological role of a bifunctional enzyme, 3,4-dihydrocoumarin hydrolase (DCH), which is capable of both hydrolysis of ester bonds and organic acid-assisted bromination of organic compounds, was investigated. Purified DCH from Acinetobacter calcoaceticus F46 catalysed dose-and time-dependent degradation of peracetic acid. The gene (dch) was cloned from the chromosomal DNA of the bacterium. The dch ORF was 831 bp long, corresponding to a protein of 272 amino acid residues, and the deduced amino acid sequence showed high similarity to those of bacterial serine esterases and perhydrolases. The dch gene was disrupted by homologous recombination on the A. calcoaceticus genome. The dch disruptant strain was more sensitive to growth inhibition by peracetic acid than the parent strain. On the other hand, the recombinant Escherichia coli cells expressing dch were more resistant to peracetic acid. A putative catalase gene was found immediately downstream of dch, and Northern blot hybridization analysis revealed that they are transcribed as part of a polycistronic mRNA. These results suggested that in vivo DCH detoxifies peroxoacids in conjunction with the catalase, i.e. peroxoacids are first hydrolysed to the corresponding acids and hydrogen peroxide by DCH, and then the resulting hydrogen peroxide is degraded by the catalase.Keywords: Acinetobacter calcoaceticus; catalase; 3,4-dihydrocoumarin hydrolase; perhydrolase; peroxoacid.In a previous study, we found and isolated a novel lactonohydrolase, 3,4-dihydrocoumarin hydrolase (DCH), from Acinetobacter calcoaceticus F46 [1]. The amino acid sequences of the N-terminal and internal peptide of DCH exhibited high similarity to those of several serine-hydrolases and perhydrolases (nonhaem haloperoxidases). Furthermore, the enzyme also showed both hydrolysis activity toward aromatic lactones, such as 3,4-dihydrocoumarin (Scheme 1), and organic acid-assisted bromination activity toward monochlorodimedon (2-chloro-5,5-dimethyl-1,3-cyclohexanedione).Perhydrolases were originally identified as the enzymes catalysing halogenation reactions of various organic compounds, because these enzymes catalyse the halogenation of various organic compounds in the presence of hydrogen peroxide, halide ions, and organic acids. Various perhydrolases have been isolated, mainly from Pseudomonas and Streptomyces species [2][3][4][5][6][7][8]. Several of these species are known to produce halogenated metabolites. Perhydrolases had been, as a result of this, thought to be involved in the synthesis of halogenated metabolites in vivo, and had been called Ônonhaem haloperoxidasesÕ, to distinguish them from the ÔtrueÕ haloperoxidases, which are haem-or vanadiumdependent enzymes [9]. However, there is now evidence that perhydrolases do not participate in the biosynthesis of halogenated compounds. A perhydrolase-deficient mutant of Pseudomonas fluorsecens yielded a chlorinated metabolite, pyrrolnitrin, like the parent strain [4], and the Streptomyces enzyme, which chlorinates tetracycline, is not relate...