A Dehydrogenase Dual Hydrogen Abstraction Mechanism Promotes Estrogen Biosynthesis: Can We Expand the Functional Annotation of the Aromatase Enzyme?

Abstract

Cytochrome P450 (CYP450) enzymes are involved in the metabolism of exogenous compounds and in the synthesis of signaling molecules. Among the latter, human aromatase (HA) promotes estrogen biosynthesis, which is a key pharmacological target against breast cancers. After decades of debate, interest in gaining a comprehensive picture of HA catalysis has been renewed by the recent discovery that compound I (Cpd I) is the reactive species of the peculiar aromatization step. Herein, for the first time, a complete atomic-level picture of all controversial steps of estrogen biosynthesis is presented. By performing cumulative quantum-classical molecular dynamics and metadynamics simulations of about 180 ps, it is revealed that the most likely enzymatic path relies on three factors: 1) androstenedione enolization and compound 0 (Cpd 0) formation through a proton network mediated by Asp309; 2) subsequent formation of Cpd I, upon rearrangement of the Asp309 side chain and the establishment of a proton network involving Asp309 and Thr310; and 3) after two hydroxylation reactions, 19,19-gem-diol is converted into estrone by Cpd I, through an uncommon dehydrogenase-like dual hydrogen abstraction mechanism. As a result, HA performs estrogen biosynthesis by merging hydroxylase with dehydrogenase activity, which suggests that the need to perform complex chemical transformations led nature to engineer HA, and possibly other steroidogenic CYP450s, by expanding its range of functions to achieve an optimal catalytic efficiency.

The Catalytic Mechanism of Steroidogenic Cytochromes P450 from All-Atom Simulations: Entwinement with Membrane Environment, Redox Partners, and Post-Transcriptional Regulation

“…In recent years, a growing body of experimental and theoretical studies have contributed to unravel controversial steps of the complex catalytic mechanisms of steroidogenic CYP450s [41,45,47,59,71,108]. Nonetheless, a comprehensive theoretical investigation of CYP17A1 and CYP11A1 catalytic mechanism is still missing in order to further confirm the functional classification of these enzymes.…”

“…Although it is well established that the reactive species for most of the CYP450 oxidations is the radical cation complex, Cpd I, an alternative mechanism, involving the ferric peroxo complex, FeO 2 − , has been at times proposed to account for atypical reactions, such as the breaking of a C-C bond ( Figure S1) [39]. Furthermore, in order to rationalize some of these unusual chemical reactions, a dual hydrogen abstraction (DHA) mechanism, relying on two subsequent hydrogen abstraction steps, has been put forward [40,41]. In this case, a first C-H abstraction is followed by a second H-abstraction from the hydroxyl group of the radical intermediate, leading to the aldehyde formation.…”

“…Conversely, recent experimental studies, based on resonance Raman spectroscopy [69], Kinetic Solvent Isotope Effect [70], and new 18 O labelling studies [71,75] definitively pinpointed Cpd I as the reactive species of the last step. In a recent work, by performing a sophisticated computational protocol relying on extensive FF-based and QM/MM metadynamics simulations, a comprehensive atomic-level picture of AR catalysis was provided (Figure 7), disclosing that, after the first two hydroxylation steps, in which also a 2,3-enol formation takes place, a non-canonical DHA mechanism completes substrate aromatization (Figure 6c) [41,72]. Thus, the mysterious third AR catalytic step may be classified within a class of non-canonical reactions promoted by CYP450s, contributing to enlarge the range of AR functions beyond that of canonical hydroxylation reactions.…”

“…(a) The sequential oxidation steps catalyzed by the aromatase (AR) enzyme. Aromatization mechanism involving (b) the ferric peroxo complex intermediate, FeO2 − , as the reactive species, and (c) a dual hydrogen abstraction mechanism[41].…”

“…(a) The relative free energy profile (kcal/mol) for the aromatization (third catalytic) step, considering the keto (R k ) and enol (R en ) form of androstenedione as a reagent. (b) Representative snapshots, as obtained in ref[41], for the reagent (R en ), intermediate (I en ) and product (P en ) states formed along the lowest energy path, are reported.…”