Integration of Styrene Oxide Isomerase into a Biocatalytic Pathway for the Synthesis of Beta-lactam Antibiotics.
Huong Trinh
Department of Chemistry & Biochemistry
Faculty Supervisor: George Gassner
We have developed a cell-free pathway for the biocatalytic synthesis of existing and novel beta-lactam antibiotics that uses styrene derivatives as starting materials. The integral membrane heme-protein styrene oxide isomerase (SOI) plays a central role in this pathway, where it catalyzes the transformation of styrene oxides to corresponding aldehyde products. SOI proceeds through a Meinwald rearrangement in which the open iron coordination sphere of the ferric heme interacts as a Lewis acid with the lone pair electrons of the oxygen atom of styrene oxide. In the present work we determine the redox potential of the heme coenzyme of SOI to be -246 mV . We further show that under anaerobic conditions that SMOB, the flavin reductase component of styrene monooxygenase (SMO) catalyzes the rapid reduction of SOI. Since ferrous heme is expected to be a weaker Lewis acid than ferric, heme reduction is expected to decrease the rate of styrene oxide isomerization. Under anoxic conditions, the SOI-bound heme may play a further role in energy recovery by shuttling surplus electrons from the reduced cytosolic FAD pool to the coenzyme Q pool periplasmic membrane. In our cell-free biocatalytic pathway, aerobic conditions are maintained, preventing the accumulation of reduced FAD and heme reduction. In the penultimate step of our cell-free biocatalytic pathway, aldehydes synthesized by SOI are oxidized to thioesters by a coenzyme A acylating aldehyde dehydrogenase. To decrease operating costs, low-molecular-weight coenzyme A surrogate molecules, N-acetylcyteamine, dithiothreitol, and dithioerythritol are used in place of coenzyme A. These coenzyme A surrogates bind to the ferric heme of SOI but do not significantly interfere with the rate of the styrene oxide isomerization reaction. This finding is consistent with the formation of relatively weak and kinetically labile heme-thiolate complexes that do not represent a significant source of competitive inhibition.