Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in the substrate specificity of the biphenyl 2,3-dioxygenases from both organisms.

Pseudomonas putda Idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50%o (vol/vol). Growth also occurs on Luria-Bertani medium in the presence of a wide range of organic solvents. The ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethylphthalate (log POCT = 2.3) being the most polar solvent tolerated. During growth with p-xylene (20%o [vol/voll), there was an initial lag period accompanied by cell death, which was followed by a period of exponential growth. The stationary phase of growth was characterized by a dramatic decrease in cell viability, although cell dry weight and turbidity measurements slowly increased. Electron micrographs revealed that during growth in the presence of p-xylene, the outer cell membrane becomes convoluted and membrane fragments are shed into the culture medium. At the same time, the cytoplasmic membrane invaginates, forming vesicles, and becomes disorganized. Electron-dense intracellular inclusions were observed in cells grown with p-xylene (20%o [vol/vol]) and p-xylene vapors, which are not present in cells grown with succinate. Attempts to demonstrate the presence of plasmid DNA in P. putida Idaho were negative. However, polarographic studies indicated that the organism utilizes the same pathway for the degradation of toluene, m-xylene, and p-xylene as that used by P. putida mt-2 which contains the TOL plasmid pWWO. Southern hybridization experiments showed that P. putida Idaho contains regions of DNA that are homologous to genes encoding enzymes of the upper operon in the TOL plasmid pWWO.

Bacterial strains expressing toluene and naphthalene dioxygenase were used to examine the sequence of reactions involved in the oxidation of 1,2-dihydronaphthalene. Toluene dioxygenase of Pseudomonas putida F39/D oxidizes 1,2-dihydronaphthalene to (؉)-cis-(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene, (؉)-(1R)-hydroxy-1,2-dihydronaphthalene, and (؉)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, naphthalene dioxygenase of Pseudomonas sp. strain NCIB 9816/11 oxidizes 1,2-dihydronaphthalene to the opposite enantiomer, (؊)-cis-(1R,2S)-dihydroxy-1,2,3,4-tetrahydronaphthalene and the identical (؉)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. Recombinant Escherichia coli strains expressing the structural genes for toluene and naphthalene dioxygenases confirmed the involvement of these enzymes in the reactions catalyzed by strains F39/D and NCIB 9816/11. 1-Hydroxy-1,2-dihydronaphthalene was not formed by strains expressing naphthalene dioxygenase. These results coupled with time course studies and deuterium labelling experiments indicate that, in addition to direct dioxygenation of the olefin, both enzymes have the ability to desaturate (dehydrogenate) 1,2-dihydronaphthalene to naphthalene, which serves as a substrate for cis dihydroxylation.Pseudomonas putida F1 and Pseudomonas sp. strain NCIB 9816-4 initiate the oxidation of toluene and naphthalene by the addition of both atoms of molecular oxygen and two hydrogen atoms to the aromatic nucleus to form (ϩ)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) (14, 21) and (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (19,20), respectively. These reactions are catalyzed by multicomponent enzyme systems designated toluene dioxygenase (TDO) (16, 31) and naphthalene dioxygenase (NDO) (13).Current interest in TDO and NDO stems from the fact that, in addition to the enantiospecific reactions shown above, both enzymes also oxidize a wide range of substrate analogs to optically active products (5,6,10,11,25). Many of these compounds have been used as chiral synthons to synthesize a number of compounds of interest to the pharmaceutical and specialty chemical industries (references 9, 10, and 17 and references cited therein).In addition to forming arene cis-dihydrodiols, the TDO expressed by strain F39/D catalyzes the monohydroxylation of indan to (1R)-indanol (7, 29). The same reaction is catalyzed by TDO expressed by P. putida UV4, a strain studied extensively by Boyd and his colleagues (5, 6). In contrast, NDO from strain NCIB 9816/11 oxidizes indan to (1S)-indanol and also catalyzes the desaturation of indan to indene. The latter compound is then oxidized by the enzyme to (1S)-indenol and cis-(1R,2S)-indandiol (15). The TDOs expressed by F39/D and UV4 do not catalyze the desaturation of indan to indene (6,29). Strain UV4 does, however, oxidize 1,2-dihydronaphthalene (compound I, Fig. 1) to (ϩ)-(1R)-hydroxy-1,2-dihydronaphthalene (compound II), (ϩ)-cis-(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene (compo...

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