Characterization of two radical S-adenosyl-L-methionine enzymes in the biosynthesis of aminoglycosides

Biosynthetic studies of natural products are essential to the discovery and development of new drugs, because by understanding biosynthetic pathways and the enzymes that characterize them, new pathways can be engineered for the production of new compounds with improved clinical properties. Aminoglycosides have traditionally been used as important antibiotics, but resistance against aminoglycosides is well known. This has resulted in a need to better understand the biosynthesis of aminoglycosides. Besides the practical value, the discovery of new enzymes with unprecedented functions in the biosynthesis of aminoglycosides can expand our knowledge and advance our understanding of enzyme catalysis. The work described in this dissertation focuses on the in vitro characterization of two radical S-adenosyl-L-methionine (SAM) enzymes in the biosynthesis of aminoglycosides. First, GenK is a cobalamin (Cbl)-dependent radical SAM enzyme that is responsible for catalyzing the methylation of gentamicin X₂ to produce G418. In vitro assays of purified and reconstituted GenK from Micromonospora echinospora showed that this enzyme is a radical SAM enzyme with one [4Fe-4S] cluster. Assays of GenK with SAM, gentamicin X₂ and Cbl confirmed that the methylation reaction occurs at an unactivated carbon during gentamicin biosynthesis. Isotope labeling experiments strongly suggested that SAM is the preliminary methyl donor to cobalamin, followed by secondary transfer from Me-Cbl to gentamicin X₂. It was demonstrated that GenK also accepts alternative aminoglycoside substrates besides gentamicin X₂. Second, three possible mechanisms for the GenK reaction were suggested and tested. Experiments investigating the stoichiometry of the GenK reaction revealed 5'-deoxyadenosine (5'-dAdo), S-adenoxylhomocysteine (SAH), and G418 were produced in equal proportion and one-to-one with each methylation reaction catalyzed by GenK. The experiment with labeled substrates indicated that the 6'-pro-R-hydrogen atom of gentamicin X₂ is abstracted by 5'-dAdo• and that methylation occurs with retention of configuration at C6'. Several substrate derivatives were synthesized to investigate the manner of methyl transfer from cobalamin to the substrate. Finally, the in vitro characterization of AprD4 and AprD3 in the biosynthesis of C3'-deoxyaminoglycoside was also conducted. Purified and reconstituted AprD4 from Streptomyces tenebrarius is a radical SAM enzyme, catalyzing homolysis of SAM to 5'-dAdo in the presence of paromamine to produce 4-oxolividamine. AprD3 from the same strain is shown to be a dehydrogenase acting as the reductase counterpart to AprD4 to catalyze the reduction of 4-oxolividamine to generate lividamine. The experiments with labeled compounds confirmed the regiochemistry of hydrogen atom abstraction by 5'-dAdo• and the stereochemical course of C3'-deoxygenation of paromamine.