Unravelling the C-C and C-N coupling mechanism for the CYP96T1-catalyzed biosynthesis of Amaryllidaceae alkaloids

Abstract

Plant cytochrome P450s (CYPs) comprise a vast family of enzymes intricately involved in the biosynthesis of natural products. However, the mechanistic understanding of the plant P450s’ catalysis is rather limited before. In this study, comprehensive simulations were performed to elucidate the C-C and C-N coupling mechanism involved in the biosynthesis of Amaryllidaceae alkaloids catalyzed by CYP96T1. Our simulations propose that the C-C coupling process follows a diradical mechanism. Especially, the proximal phenol OH group can be H-bonded to the active species of Cpd I, allowing the facile hydrogen atom transfer (HAT) from the phenol OH to Cpd I. In addition, the initial HAT disrupts the H-bond interaction between the proximal phenol group and the Cpd II species, enabling the approach of the distal phenol group for the second HAT, en route to the diradical species. The following para-para' C-C phenolic coupling reaction was observed to take place within the active site. In the context of the C-N coupling reaction, our calculations indicate that the reactions proceed via an Aza-Michael addition mechanism, facilitated by the acid-base catalysis provided by phosphate ions from the pH buffer. Notably, both the C1-N and C5-N coupling exhibit comparable energy barriers, resulting in the formation of the experimentally observed products 3 ((6R, 1S)-noroxomaritidine) and 4 ((6R, 5S)-noroxomaritidine). Our investigation has not only elucidated the mechanistic aspects of the C-C and C-N formation mechanism underlying the biosynthesis of Amaryllidaceae alkaloids, but also underscored the synergy between enzymatic transformations within P450 and non-enzymatic transformations within an aqueous solution during the biosynthesis of natural products.