Abstract 1178 Discovery and Characterization of an L-DOPA Dioxygenase from Streptomyces hygroscopicus jingganensis

The largest natural reservoir of untapped carbon can be found in the cell-wall strengthening, plant woody-tissue polymer, lignin - a polymer of catechols or 1,2-dihydroxybenzene monomers. The catecholic carbon of lignin could be valorized into feedstocks and natural products by way of catabolic and biosynthetic transformations including the oxygen-dependent cleavage reaction of extradiol dioxygenase (EDX) enzymes. The EDX L-DOPA 2,3-dioxygenase was first discovered as part of a biosynthetic gene cluster to the natural product antibiotic, lincomycin, and also contributes to the biosyntheses of anthramycin, sibiromycin, tomaymycin and hormaomycin. Using these L-DOPA 2,3-dioxygenases as a starting point, we searched sequence space in order to identify new sources of dioxygenase driven natural product diversity. A "VOC family protein" from S. hygroscopicus jingganensis was identified using bioinformatic methods and biochemically investigated for dioxygenase activity against a suite of natural and synthetic catechols. Oxygen consumption assays were used to screen and identify substrates, and a kinetic model of oxygen consumption was developed to evaluate activity of the S. hygroscopicus jingganensis VOC-family-protein with respect to activity of L-DOPA dioxygenases from S. lincolnensis and S. sclerotialus. Lastly, these data were integrated with steady-state kinetic methods to observe the formation of the EDX cleavage product with UV-Visible spectroscopy. The genomic context and enzymatic activity of the S. hygroscopicus jingganensis VOC family protein are consistent with an L-DOPA 2,3-dioxygenase contained within a cryptic biosynthetic pathway. NSF CHE 1708237 (K.L.C) and NSF CHE 1708234 (L.W.P.)