No abstract

Enzymes catalyzing [4+2] cycloaddition have attracted increasing attention because of their key roles in natural product biosynthesis. Here, we solved the X‐ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyze intramolecular [4+2] cycloadditions to form enantiomeric decalin scaffolds during biosynthesis of the HIV‐1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin. Computational modeling, using molecular dynamics simulations as well as quantum chemical calculations, demonstrates that the reactions proceed through synergetic conformational constraints assuring transition state‐like substrates folds and their stabilization by specific protein‐substrate interactions. Site‐directed mutagenesis experiments verified the binding models. Intriguingly, the flexibility of bound substrates is largely different in two enzymes, suggesting the distinctive mechanism of dynamics regulation behind these stereoselective reactions. The proposed reaction mechanism herein deepens the basic understanding how these enzymes work but also provides a guiding principle to create artificial enzymes.

Steroid hormones modulate numerous physiological processes in various higher organisms. Research on the physiology, biosynthesis, and metabolic degradation of steroid hormones is crucial for developing drugs, agrochemicals, and anthelmintics. Most steroid hormone biosynthetic pathways, excluding those in insects, have been elucidated, and the roles of several cytochrome P450s (CYPs, P450s), heme (iron protoporphyrin IX)-containing monooxygenases, have been identified. Specifically, P450s of the animal steroid hormone biosynthetic pathways and their three dimensional structures and reaction mechanisms have been extensively studied; however, the mechanisms of several uncommon P450 reactions involved in animal steroid hormone biosynthesis and structures and reaction mechanisms of various P450s involved in plant and insect steroid hormone biosynthesis remain unclear. Recently, we determined the crystal structure of P450 responsible for the first and rate-determining step in brassinosteroids biosynthesis and clarified the regio- and stereo-selectivity in the hydroxylation reaction mechanism. In this review, we have outlined the general catalytic cycle, reaction mechanism, and structure of P450s. Additionally, we have described the recent advances in research on the reaction mechanisms of steroid hormone biosynthesis-related P450s, some of which catalyze unusual P450 reactions including C–C bond cleavage reactions by utilizing either a heme–peroxo anion species or compound I as an active oxidizing species. This review article is an extended version of the Japanese article, Structure and mechanism of cytochrome P450s involved in steroid hormone biosynthesis, published in SEIBUTSU BUTSURI Vol. 61, p. 189–191 (2021).

Enzymes catalyzing [4+2] cycloadditions are involved in the formation of complex structures found in natural products, and play key roles in the control of stereochemistry. Fsa2 and Phm7 catalyze intramolecular [4+2] cycloaddition to form enantiomeric decalin scaffolds during the biosynthesis of HIV-1 integrase inhibitors, equisetin, and an opposite stereochemical homolog, phomasetin. Here, we solved the X-ray crystal structures of substrate-free Fsa2 and Phm7, and an inhibitor-bound Phm7 to understand the molecular basis underlying stereoselective cycloaddition reactions. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, which explain the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyze the stereoselective cycloaddition. This powerful combination of experimental and theoretical approaches highlights the distinct molecular mechanisms of enzyme-mediated [4+2] cycloaddition and its stereoselectivity.

Bindung einer linksdrehenden oder rechtsdrehenden Faltung mit Flip: Eine lineare Kohlenwasserstoffkette, die ein Dien und ein Dienophil enthält, wird gefaltet und durch intramolekulare [4+2]‐Cycloaddition in ein Dekalingrundgerüst umgewandelt. Die Substrate falten entgegengesetzt, binden aber in der gleichen Ausrichtung in die Taschen eines Paares von Diels‐Alderasen, um Dekalingerüste mit enantiomeren Konformationen zu liefern, wie Naoki Kato, Shunji Takahashi und Shingo Nagano im Forschungsartikel auf S. 22575 berichten.