Enzymatic activation of oxygen in the biosynthesis of polyketide antibiotics

The electron structure of molecular oxygen (O2) is such that it cannot spontaneously react with organic molecules, a feature that protects organisms from oxidative stress. However, oxygenases can insert O2 in organic substrates. The enzymes do this after activating O2 for the reaction, most often with the aid of an organic or inorganic cofactor. A number of different cofactors have been discovered in nature thus far. In this thesis, five oxygenases with three different catalytic machineries for activation of oxygen have been investigated structurally and biochemically. The enzymes participate in the biosynthesis of aromatic polyketide antibiotics. These natural products possess a range of biological activities, and a number of them have been employed as therapeutic agents as, for instance, antimicrobials and cytostatics in the treatment of cancer. Presently the number of antibiotic-resistant bacteria is increasing alarmingly, and the polyketide-derived cytostatic agents are causing severe side-effects to patients. Thus development of novel polyketide antibiotics with beneficial medicinal properties is highly desirable. Structure-function studies of the biosynthetic enzymes could be of aid in this. Firstly, three flavin-dependent aromatic hydroxylases, PgaE, CabE and RdmE, were chosen as targets of study. They belong to a family of aromatic hydroxylases, for which para-hydroxybenzoate hydroxylase is a well-characterized example. Main features in the catalysis are common to all enzymes of the family and the overall structures are similar. However, there appear to be intriguing differences regarding, for instance, substrate entry and oligomeric state. With RdmE, a ternary complex was obtained with the polyketide substrate, offering insights into substrate recognition. RdmB exhibits high sequence and structural similarity to SAM-dependent smallmolecule methyl transferases, however it hydroxylates its substrate. The structural and biochemical studies revealed the enzyme to indeed utilize SAM as a cofactor in the reaction, but a substrate-carbanion species carries out the activation of O2. Subtle changes in the orientation of the substrate and the cofactor binding appear to be responsible for the change of activity from a methyl transferase to a hydroxylase. This is a novel function for SAM, and identifies RdmB as a new type of hydroxylase. SnoaB is a small cofactor-independent oxygenase. Based on structural and biochemical characterization a mechanism proceeding via a substrate carbanion intermediate is proposed. This is in accordance with recent results obtained for other cofactorindependent oxygenases. Despite the very different three-dimensional structures and the different machineries utilized, the five enzymes appear to activate molecular oxygen in a very similar manner: they all form proteinand resonance-stabilized carbanions, which form hydroperoxy-intermediates with O2. In addition, the study has provided further examples of divergent evolution, a phenomenon which appears to be a common feature in polyketide biosynthesis. Furthermore, some insights into enzymatic recognition of the large hydrophobic polyketide substrates have been obtained. LIST OF PUBLICATIONS I. Jansson, A. , Koskiniemi, H. , Erola, A. , Wang, J., Mantsala, P., Schneider, G. and Niemi, J. (2005) Aclacinomycin 10-hydroxylase is a novel substrateassisted hydroxylase requiring S-adenosyl-L-methionine as cofactor. J. Biol. Chem. 280, 3636-3644. II. Koskiniemi, H. , Metsa-Ketela, M. , Dobritzsch, D. , Kallio, P., Korhonen, H. , Mantsala, P., Schneider, G. and Niemi, J. (2007) Crystal structures of two aromatic hydroxylases involved in the early tailoring steps of angucycline biosynthesis. J. Mol. Biol. 372, 633-648. III. Lindqvist, Y. , Koskiniemi, H. , Jansson, A. , Sandalova, T. , Schnell, R. , Liu, Z. , Mantsala, P., Niemi, J. and Schneider, G. (2009) Structural basis for substrate recognition and specificity in aklavinone-11-hydroxylase from rhodomycin biosynthesis. Submitted IV. Koskiniemi, H. *, Grocholski, T. *, Schneider, G. and Niemi, J. (2009) Expression, purification and crystallization of the cofactor-independent monooxygenase SnoaB from the nogalamycin biosynthetic pathway. Acta Cryst. F65, 256-259. V. Grocholski, T. *, Koskiniemi, H. *, Lindqvist, Y. , Niemi, J. and Schneider, G. (2009) Crystal structure of SnoaB, a cofactorless monooxygenase from Streptomyces nogalater: implications for the reaction mechanism. Manuscript *Equally contributing authors A RELATED PUBLICATION NOT INCLUDED IN THE THESIS Jansson, A. *, Koskiniemi, H. *, Mantsala, P., Niemi, J. and Schneider, G. (2004) Crystal structure of a ternary complex of DnrK, a methyltransferase in daunorubicin biosynthesis, with bound products. J. Biol. Chem. 279, 41149-41156. *Equally contributing authors