Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe 2 S 2 centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of K mA ‫؍‬ 9 ؎ 1 M, k cat ‫؍‬ 3.9 ؎ 0.2 s ؊1 , and K mO 2 ‫؍‬ 16 ؎ 2 M (100 mM sodium phosphate, pH 7.0; 25°C), where K mO 2 represents the K m for O 2 and K mA represents the K m for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate Ӎ 3-chlorobenzoate > p-toluate Ӎ 4-chlorobenzoate ӷ o-toluate Ӎ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O 2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O 2 utilization, with >10 times more O 2 being consumed than benzoate. However, the apparent K m of TADO for these benzoates was >100 M, indicating that they do not effectively inhibit the turnover of good substrates.Ring-hydroxylating dioxygenases catalyze the dihydroxylation of aromatic compounds. These multicomponent enzymes are key players in the microbial degradation of aromatic compounds and thus constitute an essential link in the global carbon cycle (9, 18). Moreover, ring-hydroxylating dioxygenases are important biocatalysts in a growing number of applications: the cis-dihydrodiols typically produced by these enzymes are useful chiral synthons, and these enzymes catalyze a range of other reactions of use to green chemistry (19,28). Their versatility has also been exploited in a variety of bioremediation applications (47). Despite recent advances in the understanding of these dioxygenases, including structures of naphthalene dioxygenase (NDO) (5, 31), many important aspects of their function remain unknown.Toluate 1,2-dioxygenase (TADO) of Pseudomonas putida mt-2 (16, 39, 49) transforms meta-and para-substituted benzoates to the corresponding cis-1,2-dihydroxycyclohexadienes (Fig. 1). TADO is encoded by the xylXYZ genes, which are part of the xyl regulon found on the pWW0 plasmid involved in the degradation of xylenes and substituted toluenes (24). Transcription of the xylXYZ genes is under control of the P m promoter, which is activated by the XylS regulator and various benzoates that act as positive effectors (37). The nucleotide sequence of xylXYZ (25) indicates that TADO is a group II dioxygenase, a group that includes anthranilate, benzoate, and 2-halobenzoate dioxygenases (ANDO, BADO, and 2-HBADO, respectively) and other enzymes that were classified as class IB dioxygenases according to a previous ...

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Biphenyl dioxygenase from the psychrotolerant bacterium Pseudomonas sp. strain Cam-1 (BPDO Cam-1 ) was purified and found to have an apparent k cat for biphenyl of 1.1 ؎ 0.1 s ؊1 (mean ؎ standard deviation) at 4°C. In contrast, BPDO LB400 from the mesophile Burkholderia xenovorans LB400 had no detectable activity at this temperature. At 57°C, the half-life of the BPDO Cam-1 oxygenase was less than half that of the BPDO LB400 oxygenase. Nevertheless, BPDO Cam-1 appears to be a typical Pseudomonas pseudoalcaligenes KF707-type dioxygenase.The cold-tolerant bacterium Pseudomonas sp. strain Cam-1, previously isolated from Arctic soil, transforms polychlorinated biphenyls (PCBs) with up to four chloro substituents at 7°C at higher rates than Burkholderia xenovorans LB400, a well-characterized PCB-degrading mesophilic bacterium (2,14). At 50°C, PCB removal by Cam-1 was diminished, but that by LB400 was not (14). The degradation of PCBs by these two strains is further distinguished by their respective congener preferences. LB400 preferentially degrades ortho-substituted PCBs with up to six chloro substituents (6). In contrast, the congener preference of Cam-1 is similar to that of Pseudomonas pseudoalcaligenes KF707, preferentially degrading parasubstituted PCBs with up to four chloro substituents (6). A third well-characterized mesophilic PCB-degrading bacterium, Pandoraea pnomenusa B-356 (previously Comamonas testosteroni B-356), preferentially transforms meta-substituted congeners with up to four chloro substituents (1).The first enzyme of the biphenyl/PCB-degrading pathway, biphenyl dioxygenase (BPDO), is one of the major determinants of the aerobic PCB-degrading capabilities of bacteria (17). Accordingly, the PCB-degrading capabilities of strains LB400 and B-356 largely reflect those of BPDO LB400 and BPDO B356 , respectively (7). BPDO is a three-component dioxygenase comprising a reductase, a ferredoxin, and an oxygenase component (iron-sulfur protein [ISP]) of ␣ 3 ␤ 3 constitution. The ␣-subunit contains a Rieske FeS cluster and, within the active site, a mononuclear iron center. BPDO utilizes NADH to activate O 2 and catalyze the 2,3-dihydroxylation of biphenyl. A crystal structure of ISP B356 , the oxygenase component of BPDO B356 , in complex with 2,6-dichlorobiphenyl has provided insight into the determinants of congener preference in BPDOs (7). For instance, Phe336 of ISP LB400 decreases the preference of this enzyme for di-para-substituted congeners (7). Moreover, BPDOs have been engineered to improve their PCB-degrading capabilities (3, 5, 18).The oxygenase component of BPDO Cam-1 shares 99%, 95%, and 70% amino acid sequence identities with those of BPDO KF707 , BPDO LB400 , and BPDO B356 , respectively. Indeed, the ␣-subunits of ISP Cam-1 and ISP KF707 differ by a single residue at position 178 (Ala in ISP Cam-1 and Val in ISP KF707 ) (13). To better understand the differences between the various BPDOs and their influence on the temperature dependence of PCB degradation by the parent strains (14), BPDO Ca...