Characterization of Cobalamin-Dependent Radical S-Adenosyl-L-Methionine Methyltransferases Involved in Antibiotic Biosynthesis

Kylie Allen

Due to increasing antibiotic resistance among dangerous pathogens, there is a critical need for new antibiotics. Enzymes represent a powerful tool for antibiotic production and discovery, however more enzymatic mechanisms must first be understood. This dissertation characterizes three members of an emerging group of enzymes known as cobalamin (Cbl)-dependent radical S-adenosyl-L-methionine (SAM) (RS) methyltransferases. RS enzymes utilize a four-iron, four-sulfur [4Fe-4S] cluster and SAM to perform difficult radical chemistry. The methyl group donor in reactions catalyzed by these enzymes is thought to be the vitamin B12-derivative methylcobalamin (CH3-Cbl(III)). These enzymes are uniquely suited for drug production processes due to their ability to perform chemistry at otherwise unreactive sites. Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. The putative Cbl-dependent RS methyltransferases Fom3 and PhpK are found in the biosynthetic pathways for two such compounds, fosfomycin and phosphinothricin, respectively. Fom3 is proposed to catalyze the methylation of an unactivated sp3-hybridized carbon, while PhpK catalyzes the P-methylation of a phosphinate substrate to form the only naturally-occurring C-P-C bond sequence. A third enzyme of interest, SD_1168, has an unknown biological function but unexpectedly catalyzes a P-methylation reaction similar to PhpK. Herein, we describe an overexpression, refolding, and reconstitution procedure that produces significant quantities of each enzyme as soluble recombinant protein. Using electron paramagnetic resonance spectroscopy, we show that these Cbl-dependent RS methyltransferases contain a RS-characteristic [4Fe-4S] cluster which transitions between a +2 "resting" state and a +1 catalytic state. Using nuclear magnetic resonance spectroscopy, we demonstrate the in vitro methylation activities of Fom3, PhpK, and SD_1168. These enzymes apparently require SAM, a strong reducing agent to produce the [4Fe-4S]+1 cluster, and CH3-Cbl(III) for activity. We demonstrate that using titanium(III) citrate as a reductant instead of sodium dithionite produces increased SD_1168 activity. Experiments with PhpK and SD_1168 suggest that Cbl functions as a coenzyme which is regenerated using a methyl group from a source other than CH3-Cbl(III). This use of Cbl is reminiscent of well-characterized Cbl-dependent methyltransferases such as methionine synthase. These studies provide the foundation necessary for the future use of Cbl-dependent RS methyltransferases for antibiotic production and discovery.

Characterization of Cobalamin-Dependent Radical S-Adenosyl-L-Methionine Methyltransferases Involved in Antibiotic Biosynthesis

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