It takes two to tango - The case of thebaine 6-O-demethylase

Publication year: 2020
Authors: Kachhap S. a*, Wojdyla Z. a*, Komorek P. a, Kluza A. a, Kurpiewska K. ab, Jachimska B. a,Borowski T.a
a - Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239 Krakow, Poland
b - Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, PL-30387 Krakow, Poland
Published in: nternational Journal of Biological Macromolecules, 2020, Vol. 163, p. 718-729
doi: 10.1016/j.ijbiomac.2020.07.030

Thebaine 6-O-demethylase (T6ODM) is an Fe(II)/2-oxoglutarate-dependent dioxygenase catalysing two oxidative O-demethylation reactions in morphine biosynthesis. Its crystal structure revealed a large active site pocket which is at least two times larger than necessary to accommodate a substrate (thebaine or oripavine) molecule. Since so far no crystal structures have been obtained for enzyme-substrate complex, which is necessary to explain the enzyme regiospecificity towards the C6-bound methoxy group, in this work we used computational methods and multi-parametric surface plasmon resonance measurements to elucidate the most likely structure of this complex and the reaction mechanism starting therefrom. Results of simulations and experiments unanimously indicate that the enzyme-substrate complex of T6ODM has a 1:2 stoichiometry. The key residues responsible for substrate binding are: Val-128, Glu-133, Met-150 and Agr-219 for the substrate in the distal position, and Asp-144, Leu-235 and Leu-353 for the proximal substrate molecule. QM/MM and DFT calculations show that the oxo ligand is bound trans to His-295 and the enzyme catalyzes hydroxylation of the C6-bound methoxy group according to the established rebound mechanism. The final stage of the demethylation reaction, which includes deformylation and enol-keton tautomerization steps, is most likely catalysed by water molecules and takes place in the solvent.


MP-SPR keywords: adsorbed mass, enzyme, molecular dynamic simulations, molecular dynamics (MD) simulations, small molecular weight drugs