Thesis advisor: Steven D. Bruner / Thesis advisor: Mary Roberts / The dioxygenase DpgC performs a key step in the biosynthesis of 3,5-dihydroxyphenylglycine (DPG), a nonproteogenic amino acid found in the vancomycin family of antibiotics. Remarkably, DpgC performs a 4-electron oxidation without the use of metals or cofactors. The tools of synthetic organic chemistry, enzymology and structural biology were used to study this enzyme. We have solved the first structure of an enzyme of this oxygenase class, in complex with a bound substrate mimic. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is located adjacent to the site of oxidation on the substrate. The use of a designed, synthetic substrate analog allowed us to gain unique insights into the chemistry of oxygen activation. We systematically probed the importance of active site residues by engineering conservative changes using site-directed mutagenesis. The kinetic parameters of these constructs imply that the phenolic hydroxyls of the substrate are of particular importance. These conclusions were verified by kinetic evaluation of synthetic substrate analogs. We have synthesized cyclopropyl substrate derivatives to probe the electron transfer step. The single electron oxidation should produce a radical species capable of opening the cycloproyl ring, thus providing a handle of detection. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Enzymes have found applications in a broad range of industrial production processes. While high catalytic activity, selectivity and mild reaction conditions are attractive advantages of the biocatalysts, particularly costs arising from required cofactors pose a sever limitation. While cofactor-recycling systems are available, their use implies constraints for process set-up and conditions, which are a particular problem e.g. for solid-gas-phase reactions. Several oxidoreductases are able to directly exchange electrons with electrodes. Hence, the co-immobilization of both, an electron-utilizing and an electron-generating oxidoreductase on conductive nanoparticles should facilitate the direct electron flow from an enzymatic oxidation to a reduction reaction circumventing redox-cofactors requirements. In such a set-up, hydrogenases could generate and provide electrons directly form gaseous hydrogen. This thesis describes the co-immobilization of the oxygen tolerant hydrogenases from C. eutropha or C. metallidurans and cytochrome P450BM3 as test system. Conductive material in the form of carbon nanotubes (CNT) serves as a suitable support. A combination of the hydrogenase and the catalytic domain of P450BM3 immobilized on carbon nanotubes were tested for the oxidation of lauric acid in the presence of hydrogen instead of an electron-transport cofactor. The GC-MS analysis reveals the conversion of 4% of lauric acid (LA) into three products, which correspond to the hydroxylated lauric acid in three different positions with a total turnover (TON) of 34. The product distribution is similar to that obtained when using the wildtype P450BM3 with the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. Such electronic coupling couldn’t be achieved for the conversion of other substrates such as propane and cyclohexane, probably due to the high uncoupling rate within the heme-domain of cytochrome P450BM3 when unnatural substrates are introduced.