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Abstract

Dihydroorotate dehydrogenase (DHODH), which catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, is a validated therapeutic target in cancer, autoimmune disorders and infectious diseases. DHODH is hypothesized to utilize a ping-pong catalytic mechanism. However, available crystal structures of DHODH in complex with small molecule inhibitors are irreconcilable with this model, showing simultaneous occupancy of both substrate binding sites. To elucidate the structural basis for DHODH-mediated catalysis, we resolved the structures of two DHODH holoenzymes. These structures capture novel conformational states that show mutually exclusive substrate binding. The holoenzyme structures also suggest that conformational changes in the catalytic loop of DHODH play a critical role in regulating enzyme activity. To map the functional landscape underlying DHODH inhibitor resistance, we performed deep mutational scanning drug-resistance screens with two clinically-relevant structurally distinct DHODH inhibitors, BAY2402234 and brequinar. Resistance variants cluster in the inhibitor binding site and at a previously unappreciated surface pocket on DHODH that allosterically regulates ubiquinone binding. Together, our findings provide structural evidence for the ping pong model of catalysis, define the mutational landscape governing inhibitor resistance, and reveal novel structural vulnerabilities in DHODH that can be utilized for future drug development.

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