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The mechanism of the non-enzymatic breakdown of the tetrahedral intermediate of MurA (a carboxyvinyl transferase) was determined in order to better understand the corresponding enzyme catalyzed reaction. The breakdown was general acid-catalyzed, with simultaneous C-O bond cleavage and protonation at the bridging oxygen of the phosphate leaving group. The carboxyl was shown to play an important role in the breakdown, with its pKₐ of 3.2 appearing in the rate 𝘷𝘦𝘳𝘴𝘶𝘴 pH profile. The product distribution varied with pH. At pH 1, the products were predominantly UDP-GIcNAc, pyruvate and phosphate. Increasing proportions of MurA ketal arising from intramolecular attack of the 4'-0H, and EP-UDP-GicNAc resulting from elimination were observed with higher pHs. The MurA ketal was structurally characterized, and shown to be analogous to the ketal formed by AroA, a related enzyme. pKₐs corresponding to those expected of phosphate monoesters (1.6 and 6.2) were found in the product distribution 𝘷𝘦𝘳𝘴𝘶𝘴 pH profile. Observation of these pKₐs supported the initial product of phosphate departure being an oxocarbenium ion-phosphate tight ion pair complex. A corresponding oxocarbenium ion intermediate in the enzymatic reaction could not be demonstrated. / Thesis / Master of Science (MSc)

Resistance to the tetracycline antibiotics occurs primarily by efflux and ribosome protection mechanisms, however a tetracycline inactivating enzyme, TetX, was identified 15 years ago, although little is known about this mechanism. The gene encoding this enzyme was identified on a 𝘉𝘢𝘤𝘵𝘦𝘳𝘰𝘪𝘥𝘦𝘴 transposon and results from DNA sequence analysis and studies of bacterial culture media suggested that 𝘵𝘦𝘵𝘟's protein product might be a NADPH-requiring oxidase (𝘚𝘱𝘦𝘦𝘳 𝘉𝘚 𝘢𝘯𝘥 𝘚𝘢𝘭𝘺𝘦𝘳𝘴 𝘈𝘈. 𝘑 𝘉𝘢𝘤𝘵𝘦𝘳𝘪𝘢𝘭. 171(1): 148-153, 1989). We have expressed a copy of 𝘵𝘦𝘵𝘹 gene, 𝘵𝘦𝘵𝘹2, in Escherichia coli, and purified the enzyme to high purity. We showed that TetX2 is a monomeric 44 kDa cytoplasmic protein and UV-Vis and HPLC studies established that TetX contained an FAD cofactor. Continuous and stopped enzyme assays have been developed and established that that the enzyme requires 0₂ and NADPH for tetracycline degradation. Liquid chromatographic mass spectrometry (LC -MS) analysis of TetX reaction products using oxytetracycline (461 Da) as a substrate indicated that the enzyme catalyses the incorporation of one oxygen atom into oxytetracycline, resulting in a compound of 477 Da with no antibiotic activity. Steady state kinetic analysis demonstrated that TetX2 has a broad substrate specificity with the capacity to inactivate several members of the tetracycline family tested. Identification of the inactivated tetracycline product revealed that the tetracycline inactivation process is a TetX2 catalyzes tetracycline oxidation reaction. These studies provide the first biochemical analysis of a tetracycline inactivating enzyme. / Thesis / Master of Science (MSc)

Isobutylamine N-hydroxylase (IBAH) and flavin reductase (FRED) from Streptomyces viridifaciens are part of a two-component flavin-dependent monooxygenase enzyme system that catalyze the conversion of isobutylamine (IBA) to isobutylhydroxylamine (IBHA), a key step in the formation of valanimycin, an azoxy antibiotic. In this work, we present the over-expression, purification and biochemical characterization of this two-component enzyme system. IBAH and FRED were expressed and purified to homogeneity as separate proteins. FRED exhibited the oxidoreductase activity by catalyzing the oxidation of NADPH. The hydroxylation activity of IBAH was confirmed using liquid chromatography – mass spectrometry (LC-MS). Steady state kinetic data showed an oxidation activity of the monooxygenase component which proceeded at 1.97 ± 0.06 s⁻¹ as measured from oxygen consumption and in product formation, the rate was 0.012 ± 0.001 s⁻¹ , suggesting a high degree of uncoupling between product formation and oxygen consumption. In pre-steady state kinetic characterization studies, the FRED-catalyzed reduction of FAD by NADPH occurred at a rate of 10.0 ± 0.2 s⁻¹ and the KM was 490 ± 40 µM. The rate of reduction was ~1.5-fold decreased in the presence of substrate IBA whiles the KM was 500 ± 50 µM. NADH showed a markedly reduced rate of reduction with a kred of 0.34 ± 0.03 s⁻¹ with an apparent KM of 3000 ± 500 µM. The rate of flavin re-oxidation in the absence of monooxygenase IBAH was 4.79 × 10⁻⁹ M-1 s⁻¹. Our results suggest a reaction mechanism for the IBAH monooxygenase system controlled by the oxidation half reaction that may be modulated by a complex formation between the reductase and monooxygenase components. / Master of Science in Life Sciences

La rétinol déshydrogénase 8 (RDH8) est impliquée dans le cycle visuel où elle réduit le tout-trans rétinal en tout-trans rétinol, ce qui prévient l’accumulation de lipofuscine (A2E), une molécule toxique qui peut mener à la dégénérescence maculaire liée à l’âge. Il y a malgré tout une accumulation d’A2E dans les photorécepteurs et l’épithélium pigmentaire rétinien, ce qui suscite des interrogations à propos de son efficacité enzymatique. Il est donc important de caractériser ses propriétés enzymatiques pour clarifier cette question. La RDH8 est insoluble dans le lysat bactérien suite à sa surexpression dans un système E. coli. Il est cependant possible de la solubiliser en utilisant le détergent SDS, ce qui résulte en la production d’une protéine inactive et empêche ainsi la caractérisation de son activité enzymatique. Différentes approches ont donc été utilisées pour produire la RDH8 en absence de détergent. La première approche comprend l’ajout d’un ligand ou d’un cofacteur à la RDH8 lors de la surexpression et de la lyse cellulaire, afin de la rendre plus soluble. La deuxième approche consiste à utiliser la technologie des protéines de fusion afin d’obtenir une forme soluble de la RDH8 en fusion avec les étiquettes poly-His, MBP, TagR-TagR, NusA ou SUMO. La dernière approche comporte l’utilisation du système d’expression L. lactis pour permettre la sécrétion de la RDH8 dans le milieu de culture. Il a été impossible d’obtenir une forme soluble et pure de la RDH8 en raison de sa faible solubilité, de la protéolyse observée lors de la production des différentes protéines de fusion, ainsi que de l’absence d’expression dans le système L. lactis. La surexpression de la RDH8 en fusion avec différentes étiquettes dans les cellules eucaryotes Pichia pastoris pourrait toutefois permettre l’obtention d’une forme soluble de la RDH8 et la caractérisation de son activité enzymatique.