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.

Many microbial secondary metabolites are produced by multienzyme complexes comprising non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). The ketosynthase (KS) domains of polyketide synthase normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain. However, the terminal KS domain in tenuazonic acid synthetase 1 (TAS1) from the fungus Pyricularia oryzae conducts substrate cyclization. Here, we report on the unique features of the KS domain in TAS1. We observed that this domain is monomeric, not dimeric as is typical for KSs. Analysis of a 1.68 Å-resolution crystal structure suggests that the substrate cyclization is triggered via proton abstraction from the active methylene moiety in the substrate by a catalytic His-322 residue. Additionally, we show that TAS1 KS promiscuously accepts amino acyl substrates and that this promiscuity can be increased by a single amino acid substitution in the substrate-binding pocket of the enzyme. These findings provide insight into a KS domain that accepts the amino acid–containing substrate in an NRPS–PKS hybrid enzyme and provide hints to the substrate cyclization mechanism performed by the KS domain in the biosynthesis of the mycotoxin tenuazonic acid.

Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4 Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. R190A and R81A mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg¹⁹⁰ and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM derivatives with anti-osteoclast activity.

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO₂) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

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).