Enzyme-catalyzed [4+2] cycloaddition has been proposed to be a key transformation process in various natural product biosynthetic pathways. Recently Fsa2 was found to be involved in stereospecific trans-decalin formation during the biosynthesis of equisetin, a potent HIV-1 integrase inhibitor. To understand the mechanisms by which fsa2 determines the stereochemistry of reaction products, we sought an fsa2 homologue that is involved in trans-decalin formation in the biosynthetic pathway of an enantiomerically opposite analogue, and we found phm7, which is involved in the biosynthesis of phomasetin. A decalin skeleton with an unnatural configuration was successfully constructed by gene replacement of phm7 with fsa2, thus demonstrating enzymatic control of all stereochemistry in the [4+2] cycloaddition. Our findings highlight enzyme-catalyzed [4+2] cycloaddition as a stereochemically divergent step in natural product biosynthetic pathways and open new avenues for generating derivatives with different stereochemistry.

Prenylated indole alkaloid okaramines selectively target insect glutamate-gated chloride channels (GluCls). Because of their highly complex structures, including azocine and azetidine rings, total synthesis of okaramine A or B has not been achieved, preventing evaluation of the biological activities of okaramines. Biosynthetic approaches provide alternatives to accessing structurally diverse derivatives and enabling the elucidation of structure-activity relationships. To explore the biosynthetic potential of okaramines, gene knockout experiments of an okaramine-producer fungus were performed. The deletion mutants of the oxygenase genes okaB, okaD, okaE, and okaG provided analogues that were unlikely to be accumulated in the normal biosynthetic process of the wild-type strain. Analysis of the structure-activity relationships of okaramines collected from the fungal cultures revealed that 1,4-dihydroazocine and N-aliphatic group attached to the indole were crucial for GluCl-activating activity. This provided insights into further derivatization of the complex structure of okaramines in order to facilitate the development of new insecticides.

Enzyme-catalyzed [4+ +2] cycloaddition has been proposed to be akey transformation process in various natural product biosynthetic pathways. Recently Fsa2 was found to be involved in stereospecific trans-decalin formation during the biosynthesis of equisetin, apotent HIV-1 integrase inhibitor.T o understand the mechanisms by which fsa2 determines the stereochemistry of reaction products,w es ought an fsa2 homologue that is involved in trans-decalin formation in the biosynthetic pathway of an enantiomerically opposite analogue,a nd we found phm7, which is involved in the biosynthesis of phomasetin. Ad ecalin skeleton with an unnatural configuration was successfully constructed by gene replacement of phm7 with fsa2, thus demonstrating enzymatic control of all stereochemistry in the [4+ +2] cycloaddition. Our findings highlight enzyme-catalyzed [4+ +2] cycloaddition as as tereochemically divergent step in natural product biosynthetic pathwaysa nd open new avenues for generating derivatives with different stereochemistry.

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.

Natural drimane-type sesquiterpenes, including drimenol, display diverse biological activities. These active compounds are distributed in plants and fungi; however, their accumulation in bacteria remains unknown. Consequently, bacterial drimane-type sesquiterpene synthases remain to be characterized. Here, we report five drimenol synthases (DMSs) of marine bacterial origin, all belonging to the haloacid dehalogenase (HAD)-like hydrolase superfamily with the conserved DDxxE motif typical of class I terpene synthases and the DxDTT motif found in class II diterpene synthases. They catalyze two continuous reactions: the cyclization of farnesyl pyrophosphate (FPP) into drimenyl pyrophosphate and dephosphorylation of drimenyl pyrophosphate into drimenol. Protein structure modeling of the characterized Aquimarina spongiae DMS (AsDMS) suggests that the FPP substrate is located within the interdomain created by the DDxxE motif of N-domain and DxDTT motif of C-domain. Biochemical analysis revealed two aspartate residues of the DDxxE motif that might contribute to the capture of the pyrophosphate moiety of FPP inside the catalytic site of AsDMS, which is essential for efficient cyclization and subsequent dephosphorylation reactions. The middle aspartate residue of the DxDTT motif is also critical for cyclization. Thus, AsDMS utilizes both motifs in the reactions. Remarkably, the unique protein architecture of AsDMS, which is characterized by the fusion of a HADlike domain (N-domain) and a terpene synthase β domain (C-domain), significantly differentiates this new enzyme. Our findings of the first examples of bacterial DMSs suggest the biosynthesis of drimane sesquiterpenes in bacteria and shed light on the divergence of the structures and functions of terpene synthases.