Enzymes involved in the bacterial degradation of benzoate

The objective of this doctoral research project is to investigate the specificity, and the structural determinants thereof in two important classes of enzymes that catalyze successive transformations in the aerobic catabolism of aromatic compounds. The first class is ring-hydroxylating dioxygenases represented by toluate 1,2-dioxygenase of Pseudomonas putida mt-2 (TADOmt2, E.C. 1.14.12.-) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADPI, E.C. 1.14.12.10). The second class is aryl cis-diol dehydrogenases represented by l,2-cw-dihydroxy-3-methyl-cyclohexa-3,5- diene-carboxylate (m-toluate 1,2-diol) dehydrogenase from P. putida mt-2 (XylLmt2, E.C. 1.3.1.59) and l,2-cis-dihydroxy-3-phenyl-cyclohexa-3,5-diene (biphenyl 2,3-dihydrodiol) dehydrogenase from Burkholderia sp. strain LB400 (BphBLB4oo, E.C. 1.3.1.56). TADOmt2 and BADOADPI are two-component enzymes that share 63% sequence identity, but that catalyze the 1,2-dihydroxylation of different ranges of substituted benzoates. The active site of these enzymes is contained in an oxygenase of α3β3 configuration. The two components of TADOmt2 and BADOADPI were hyperexpressed, anaerobically purified and their substrate specificities were compared. Reconstituted TADOmt2 had a specific activity of 3.8 U/mg using m-toluate, and that of BADOADPI using benzoate was 5.0 U/mg. Each component had a full complement of their respective [2Fe-2S] centres. Steady-state kinetics data obtained using an oxygraph assay and by varying substrate and dioxygen concentrations were analyzed using a compulsory order ternary complex mechanism. TADOmt2 had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, kcat = 3.9 ± 0.2 s-1, and Kmo2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0, 25 °C). The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ~ 3-chlorobenzoate > p-toluate ~ 4- chlorobenzoate » o-toluate ~ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O2-utilization and yielded the corresponding 1,2-cis dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with > 10 times more O2 being consumed than benzoate. However, the apparent Km of TADOmt2 for these benzoates was > 100 μM, indicating that they do not effectively inhibit the turnover of good substrates. Reconstituted BADOADPI had greatest specificity for benzoate, displaying apparent parameters of KmA = 26±1 μM, kcat = 8.6 ±0.1 s-1, and Kmo2 = 53 ± 2 μM. The enzyme had a significantly narrower apparent specificity than TADOmt2, utilizing only four of the seven tested benzoates in the following order of apparent specificity: benzoate > m-toluate > 3-chlorobenzoate > otoluate. To investigate the structural determinants of substrate specificity in these dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were constructed and anaerobically purified. The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADPI , the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αBβT hybrid oxygenase differed slightly to that of TADOmt2 (3-chlorobenzoate > m-toluate > benzoate ~ p-toluate > 4-chlorobenzoate » o-toluate > 2-chlorobenzoate). Moreover, the αBβT hybrid catalyzed the 1,6-dihydroxylation of otoluate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho substituted benzoate was much better coupled to (Vutilization in the hybrid than in the parent. Overall, these results support the notion that the a subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenase. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function. XylLmt2 and BphBLB4oo catalyze the NAD+-dependent dehydrogenation of cisdiols in the aerobic catabolism of toluates and biphenyl, respectively. Each enzyme was heterologously expressed and purified, and their respective specificities for each of 4 aryl cis-diols were determined. Of the tested compounds, the best substrate of XylLmt2 was m-toluate 1,2-diol (apparent Km = 23.5 ± 1.6 μM and kcat = 0.31 ± 0.01 s-1) and that of BphBLB4oo was biphenyl 2,3-dihydrodiol (apparent Km = 11.2 ± 1.2 μM and kcat = 26.7 ± 0.02 s-1), consistent with the pathways in which these enzymes occur. Although the apparent specificity of BphBLB4oo for its preferred substrate was approximately 180 times higher than that of XylLmt2 for its preferred substrate, the former had a markedly narrower substrate specificity. Thus, the apparent specificity constant of XylLmt2 for 1,2- cis-dihydroxy-3-methyl-cyclohexa-3,5-diene (toluene 2,3-dihydrodiol), a noncarboxylated aryl cis-diol, was half that for m-toluate 1,2-diol. In contrast, BphBLB400 did not transform either of the tested carboxylated aryl cis-diols. Phylogenetic studies of aryl cis-diol dehydrogenases reveal that there are at least three evolutionarily distinct types of these enzymes. BphBLB400 and XylLmt2 represent separate classes of the type II (SDR-type) enzymes.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat