Mechanistic Insights into the P450 TleB-Catalyzed Unusual Intramolecular C-N Bond Formation Involved in the Biosynthesis of Indolactam V.

Indolactam V, a known biosynthetic precursor of indolactam alkaloids, is the main pharmacophore of this family and exhibits potential protein kinase C activation. A key step in the biosynthesis of indolactam V is the formation of an indole-fused nine-membered lactam core by intramolecular C-N bond formation. In this work, we report a computational study of the unique cytochrome P450 TleB enzyme-catalyzed direct and selective C-H bond amination reaction that can generate indolactam V from the dipeptide -methylvalyl-tryptophanol. By performing molecular dynamics simulations and quantum-mechanical/molecular-mechanical calculations, we revealed that the C-H bond amination involves one step of proton transfer from N1-H of the indole ring to the Fe═O unit, one step of hydrogen abstraction of N13-H in the side chain of the substrate by the Fe-OH unit, and diradical coupling, in which two conformational changes of the side chain of the substrate are necessary. In the enzyme-substrate complex of TleB, the N-H bond of the indole ring of the substrate forms a strong hydrogen bond with the Fe═O unit in compound I, and the porphyrin radical cation accepts an electron from the substrate to form the closed-shell electronic configuration. Thus, compound I in the enzyme-substrate complex cannot be described as Fe═O coupled to a porphyrin radical cation, which is different from those of other P450 enzymes. Besides, two stages of conformational changes of the side chains of the substrate may increase the relative energies of reaction intermediates by 10-12 kcal/mol. From the structure point of view, it is the rotatable long side chain of the substrate and the large flexible active pocket of TleB that make the intramolecular diradical coupling feasible. Our findings may provide useful information to further understand the Tleb-catalyzed intramolecular C-H bond amination and the other bio-catalyzed intramolecular diradical coupling.