Kinetic and structural studies of Mycobacterium tuberculosis dihydroorotate dehydrogenase reveal new insights to class 2 DHODH inhibition.

The enzyme dihydroorotate dehydrogenase (DHODH) present in Mycobacterium tuberculosis1 (MtDHODH) is a  promising target against tuberculosis. MtDHODH obtained after purification was pure with a yield of 12 mg/L of cell culture. The full kinetic characterization was performed with the substrates (dihydroorotate (DHO) and menadione (K3) with KmDHO = 258 ± 17 uM, KmK3 = 145 ± 10 uM, Kcat = 2.69 ± 0.08 (s-1). Unexpectedly, the coenzyme Q0 ( 2,3-dimethoxy-5-methyl-1,4-benzoquinone) was found to inhibit MtDHODH (Ki (Q0) = 138 ± 31 μM). To the best of our knowledge, Q0 is the first dihydroorotate-competitive inhibitor for class 2 DHODHs ever described. The structure of the full MtDHODH was solved by x-ray crystallography and molecular dynamics simulations, along with in silico solvent mapping, allow us to identify that the dynamics of the N-terminal region have a significant impact on the conformation of binding sites for drug-like molecules.