Pseudomonas sp. strain 9816-4 grows with naphthalene as the sole source of carbon and energy (9). The initial reaction is catalyzed by a multicomponent enzyme system designated naphthalene dioxygenase (NDO) (11,12,23,24). NDO catalyzes the NAD(P)H-dependent enantiospecific incorporation of dioxygen into naphthalene to form (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (26, 27) ( Fig. 1). An analogous reaction is catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1, where enantiomerically pure (ϩ)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) is the first detectable oxidation product (17,31,60). TDO also catalyzes the enantiospecific oxidation of naphthalene to (ϩ)-cis-naphthalene dihydrodiol (18,39).In addition to the enantiospecific oxidation of naphthalene and toluene, NDO and TDO from the above strains oxidize many related aromatic compounds to optically active dihydrodiols (10,18,28,30). Other bacterial dioxygenases show similar properties, and more than 130 chiral arene cis-dihydrodiols have been produced from a small number of strains (7,35,48). The high enantiomeric purity of these compounds has led to their use as chiral synthons in the enantiospecific synthesis of a wide variety of biologically active natural products (7,8,46,57). The present studies focus on another facet of this interesting group of dioxygenases, that is, their ability to catalyze reactions other than the formation of arene cis-dihydrodiols. For example, the TDO expressed by P. putida F39/D oxidizes indan to (1R)-indanol and oxidizes indene to cis-(1S,2R)-indandiol and (1S)-indenol (55). Similar reactions have been reported for TDO from P. putida UV4, although the 1-indenol produced by this strain is the (1R)-enantiomer (3, 5).We now report the identification and absolute stereochemistry of the products formed from indan and indene by NDO from Pseudomonas sp. strain 9816-4 and confirm earlier observations on the desaturation of indan to indene by NDO (22).
MATERIALS AND METHODSOrganisms. Pseudomonas sp. strain 9816/11 is a mutant which oxidizes naphthalene stoichiometrically to (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (40). This organism is a derivative of Pseudomonas sp. strain 9816-4 (9, 59), which harbors the genes for naphthalene catabolism on an 83-kb NAH plasmid designated pDTG1 (45). Pseudomonas sp. strain 9816/C84, a cured strain, was used as a control in experiments with strain 9816/11. Escherichia coli strain JM109 (DE3)[pDTG141] contains the structural genes (nahAaAbAcAd) for NDO in plasmid pT7-5 (50). Expression of NDO in this strain is inducible by the addition of isopropylthiogalactopyranoside (IPTG). E. coli JM109(DE3)[pT7-5] was used as a control in experiments with strain JM109(DE3) [pDTG141].Biotransformation experiments. Strain 9816/11 was grown at 30ЊC in mineral salts basal medium (MSB) (49) with 0.2% (wt/vol) pyruvate as a carbon source in the presence of 0.05% (wt/vol) salicylate or anthranilate. These aromatic acids induce the s...
