Mechanistic studies to enable rational design of isobutylamine N-hydroxylase.

With the emergence of bacterial resistance, identification of new diseases, and the need for new therapeutics with different efficacies, our ability to design drugs to battle bacterial infections is becoming a more urgent priority. Natural products are often useful as therapeutics for humans, though problems such as side effects and production difficulties can preclude their successful development. This proposal seeks to enable development of antibiotics through synthetic biology methods, in which the molecule?s biosynthetic pathway is engineered in order to alter the product. The Class D flavin monooxygenases are found in numerous natural product biosynthetic pathways, including that of the antibiotics valanimycin and everninomycin. With this research we hope to make the Class D flavin monooxygenases of these biosynthetic pathways amenable to engineering for synthetic biology purposes. The enzyme-catalyzed step of interest here is a biosynthetic step common to multiple antibiotics ? flavin-dependent hydroxylation of a primary amine. The enzyme responsible for this step in the valanimycin biosynthetic pathway will be biochemically characterized using transient-state kinetics. Site-directed mutagenesis of active site residues will be combined with enzymatic activity and binding studies to validate mechanistic steps and substrate binding interactions. The effect of changes in the substrate binding site on the kinetics of intermediate formation will be investigated for use in validating modifications to the enzyme?s substrate specificity. The data yielded will enable rational design of vlmH to alter its substrate binding preferences. Similar studies can be applied to other enzymes of the pathway to introduce diversity into the valanimycin final structure. Given enough time and research, this molecule could be developed into a useful therapeutic.