Crystallization and Functional Characterization of Bacterial Monooxygenase

In nature monooxygenases are enzymes which has important catalytic role in many metabolic reactions. These enzymes have ability of oxidation of C-H bonds – even unreactive ones. As these enzymes are included in many metabolic pathways, they have been targets of interest for biochemical and biotechnological purposes. Focus of this thesis is one of the recently discovered monooxygenase from Gram-negative bacterium Acinetobacter radioresistens. The studied monooxygenase conducts Baeyer-Villiger oxidation so it is called Ar-BVMO (Acinetobacter radioresistens Baeyer-Villiger monooxygenase). Ar-BVMO is Type I BVMO with Flavin prosthetic group and uses NADPH as electron donor. This monooxygenase oxidizes linear and cyclic ketones to corresponding ester and lactones. Beside normal substrates, Ar-BVMO can oxidize drugs and antibiotics that are typical substrates of the hFMO3 (human flavin-containing monooxygenases 3) which can lead to bacterial antibiotic resistance. First part of the project was focused on transformation, expression and purification of the protein. For purification two different protocols consisting of two chromatography steps (Ni affinity and size exclusion chromatography) were optimized and applied. Results of purifications led to productions of two populations of protein (oligomeric and monomeric) using first protocol, optimization of second protocol for more stable and pure protein for crystallization attempts. Aim of the second part was on crystallization trials of obtained protein. JBScreen I and JBScreen II screening kits provided by Jena Bioscience, Ecoscreen I, Ecoscreen II and Multi Xtal Screen crystallization kits provided Molecular Dimensions kits were used for screening. Screening attempts were repeated twice with and without using cofactor of the protein. Focus of the last parts of project was activity comparison of fractions obtained from different protocols and substrate determination of the enzyme using NADPH consumption assay and High Performance Liquid Chromatography (HPLC) method. Data observed suggested that different natural toxins and drugs can be oxidized by Ar-BVMO.