METABOLISM OF CHLOROTOLUENES BY BURKHOLDERIA SP STRAIN PS12 AND TOLUENE DIOXYGENASE OF PSEUDOMONAS-PUTIDA F1 - EVIDENCE FOR MONOOXYGENATIONBY TOLUENE AND CHLOROBENZENE DIOXYGENASES

The degradation of toluene by Pseudomonas putida F1 and of chlorobenze nes bg Burkholderia in sp. strain PS12 is initiated by incorporation o f dioxygen into the aromatic nucleus to form cis-dihydrodihydroxybenze nes. Toluene-grown cells of P. putida F1 and 3-chlorobenzoate-grown ce lls of Burkholderia sp. strain PS12 were found to monooxygenate the si de chain of 2- and 3-chlorotoluene to the corresponding chlorobenzyl a lcohols, Further metabolism of these products was slow, and the corres ponding chlorobenzoates were usually observed as end products, whereas the 3-chlorobenzoate produced from 3-chlorotoluene in Burkholderia sp . strain PS12 was metabolized further. Escherichia coli cells containi ng the toluene dioxygenase genes from P. putida F1 oxidized 2- and 3-c hlorotoluene to the corresponding chlorobenzyl alcohols as major produ cts, demonstrating that this enzyme is responsible for the observed si de chain monooxygenation. Two methyl- and chloro-substituted 1,2-dihyd roxycyclohexadienes were formed as minor products from 2- and 3-chloro toluene, whereas a chloro- and methyl-substituted cyclohexadiene was t he only product formed front 4-chlorotoluene. The toluene dioxygenase of P. putida Fl and chlorobenzene dioxygenase from Burkholderia sp. St rain PS12 are the first enzymes described that efficiently catalyze th e oxidation of 2-chlorotoluene.