Generation of Alternate Indole Diterpene Architectures in Two Species of Aspergilli

Abstract

The significant structural diversity and potent bioactivity of the fungal indole diterpenes (IDTs) has attracted considerable interest in their biosynthesis. Although substantial skeletal diversity is generated by the action of noncanonical terpene cyclases, comparatively little is known about these enzymes, particularly those involved in the generation of the subgroup containing emindole SA and DA, which show alternate terpenoid skeletons. Here, we describe the IDT biosynthetic machinery generating these unusual IDT architectures from Aspergillus striatus and Aspergillus desertorum. The function of four putative cyclases was interrogated via heterologous expression. Two specific cyclases were identified that catalyze the formation of epimers emindole SA and DA from A. striatus and A. desertorum, respectively. These cyclases are both clustered along with all the elements required for basic IDT biosynthesis yet catalyze an unusual Markovnikov-like cyclization cascade with alternate stereochemical control. Their identification reveals that these alternate architectures are not generated by mechanistically sloppy or promiscuous enzymes, but by cyclases capable of delivering precise regio- and stereospecificities.

Class II terpene cyclases: structures, mechanisms, and engineering

“…136 Similarly, DesB and EstB1, originating from Aspergillus desertorum and Aspergillus striatus, respectively, catalyze an unusual Markovnikov-like cyclization cascade on the IDT intermediate, resulting in emindole DB and emindole SA with exceptional regio-and stereospecicities. 137 Specically, through a mechanism targeting the Re face of the C2 olen in the IDT intermediate, DesB produces emindole DA. In contrast, EstB1, which targets the Si face, leads to the formation of emindole SA.…”

Dissection of the Catalytic Mechanisms of Transmembrane Terpene Cyclases Involved in Fungal Meroterpenoid Biosynthesis

“…Recent rapid advancements in artificial intelligence‐based protein structure prediction tools, as represented by AlphaFold2, [12] have allowed the generation of high‐accuracy protein models. For example, a recent study discovered a putative substrate binding pocket of PaxB using an AlphaFold2‐generated model, and identified several amino acid residues that may be linked with product selectivity, although the importance of these residues has not been experimentally investigated [10d] . Thus, AlphaFold2 could provide an alternative and efficient approach for developing a deep understanding of the function of Pyr4‐family terpene cyclases without requiring experimental determination of their structures.…”

Hot off the Press

“…Cyclases that produce indole diterpenes have been characterised from Aspergillus striatus and Aspergillus desertorum . 18 One cyclase, DesB was shown to catalyse an unusual Markovnikov-like cyclisation cascade leading to indole diterpenes such as emindole DA 18 .…”