| 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 (BADOADP1, E.C. 1.14.12.10). The second class is aryl cis-diol dehydrogenases represented by 1,2-cis-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 1,2-cis-dihydroxy-3-phenyl-cyclohexa-3,5-diene (biphenyl 2,3-dihydrodiol) dehydrogenase from Burkholderia sp. strain LB400 (BphBLB400, E.C. 1.3.1.56).; TADOmt2 and BADOADP1 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 alpha3beta3 configuration. The two components of TADOmt2 and BADOADP1 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 BADOADP1 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 muM, kcat = 3.9 +/- 0.2 s-1 , and KmO2 = 16 +/- 2 muM (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 O 2-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 muM, indicating that they do not effectively inhibit the turnover of good substrates. Reconstituted BADOADP1 had greatest specificity for benzoate, displaying apparent parameters of KmA = 26 +/- 1 muM, kcat = 8.6 +/- 0.1 s-1, and KmO2 = 53 +/- 2 muM. (Abstract shortened by UMI.)... |
