Probing the Mechanism of <scp>l</scp>-DOPA 2,3-Dioxygenase Using Synthetic Derivatives of 3,4-Dihydroxyhydrocinnamic Acid

Extradiol dioxygenase chemistry is well-known in the degradation of natural and man-made sources of aromatic carbon; however, the extent to which biosynthetic dioxygenases like l-DOPA 2,3-dioxygenase can degrade lignin-derived aromatic carbon has not been examined. To understand if l-DOPA 2,3-dioxygenases also demonstrate a capacity or even a proclivity to cleave lignin-derived aromatic carbon into semialdehyde products, the l-DOPA 2,3-dioxygenase reaction was evaluated with derivatives of 3,4-dihydroxyhydrocinnamic acid (DHHCA), also known as hydrocaffeic acid, a catecholic example of lignin-derived carbon. DHHCA analogues, 3-(2-bromo-4,5-dihydroxyphenyl)-propanoic acid (6-bromoDHHCA, <b>4</b>), 3-(2-cyano-4,5-dihydroxyphenyl)-propanoic acid (6-cyanoDHHCA, <b>5</b>), and 3-(4,5-dihydroxy-2-nitrophenyl)-propanoic acid (6-nitroDHHCA, <b>6</b>) were synthesized, their redox potential and p<i>K</i> _a values were evaluated, and their activity as enzymatic substrates was characterized on two l-DOPA 2,3-dioxygenases from <i>S. lincolnensis</i> and <i>S. hygroscopicus</i> <i>jingganensis</i>. The results indicate that DHHCA, similar in p<i>K</i> _a values, but smaller and more readily oxidized compared to l-DOPA, is a competent substrate for both enzymes. 6-BromoDHHCA is also readily oxidized, and due to its larger size, is more readily accommodated and cleaved in the more spacious active site of the <i>S. hygroscopicus</i> <i>jingganensis</i> l-DOPA 2,3-dioxygenase versus the <i>S. lincolnensis</i> enzyme. 6-CyanoDHHCA is acidic and difficult to oxidize and its interaction was characterized by significant enzyme inactivation as observed in the presteady state. These results argue for an integrated understanding of substrate size, redox potential, and active site capacity to understand substrate-enzyme compatibility and provide actionable insights to fuel the pursuit of dioxygenase catalysts that robustly cleave 6-X-DHHCA and other synthetically modified lignin-derived catecholic monomers as starting points for functionalized semialdehydes.