Class II terpene cyclases: structures, mechanisms, and engineering.

Covering: up to July 2023Terpene cyclases (TCs) catalyze some of the most complicated reactions in nature and are responsible for creating the skeletons of more than 95 000 terpenoid natural products. The canonical TCs are divided into two classes according to their structures, functions, and mechanisms. The class II TCs mediate acid-base-initiated cyclization reactions of isoprenoid diphosphates, terpenes without diphosphates (e.g., squalene or oxidosqualene), and prenyl moieties on meroterpenes. The past twenty years witnessed the emergence of many class II TCs, their reactions and their roles in biosynthesis. Class II TCs often act as one of the first steps in the biosynthesis of biologically active natural products including the gibberellin family of phytohormones and fungal meroterpenoids. Due to their mechanisms and biocatalytic potential, TCs elicit fervent attention in the biosynthetic and organic communities and provide great enthusiasm for enzyme engineering to construct novel and bioactive molecules. To engineer and expand the structural diversities of terpenoids, it is imperative to fully understand how these enzymes generate, precisely control, and quench the reactive carbocation intermediates. In this review, we summarize class II TCs from nature, including sesquiterpene, diterpene, triterpene, and meroterpenoid cyclases as well as noncanonical class II TCs and inspect their sequences, structures, mechanisms, and structure-guided engineering studies.

Discovery, Structure, and Engineering of a cis-Geranylfarnesyl Diphosphate Synthase.

cis-Prenyltransferases (cis-PTs) catalyze the sequential head-to-tail condensation of isopentenyl diphosphate (IPP) to allylic diphosphates, producing mixed E-Z prenyl diphosphates of varying lengths; however, the specific enzymes synthesizing cis-C25 prenyl diphosphates have not been identified. Herein, we present the discovery and characterization of a cis-geranylfarnesyl diphosphate synthase (ScGFPPS) from Streptomyces clavuligerus. This enzyme demonstrates high catalytic proficiency in generating six distinct cis-polyisoprenoids, including three C25 and three C20 variants. We determined the crystal structure of ScGFPPS. Additionally, we unveil the crystal structure of nerylneryl diphosphate synthase (NNPS), known for synthesizing an all-cis-C20 polyisoprenoid. Comparative structural analysis of ScGFPPS and NNPS has identified key differences that influence product specificity. Through site-directed mutagenesis, we have identified eight single mutations that significantly refine the selectivity of ScGFPPS for cis-polyisoprenoids. Our findings not only expand the functional spectrum of cis-PTs but also provide a structural comparison strategy in cis-PTs engineering.

Discovery and biosynthesis of bacterial drimane-type sesquiterpenoids from Streptomyces clavuligerus.

Drimane-type sesquiterpenoids (DMTs) are characterized by a distinctive 6/6 bicyclic skeleton comprising the A and B rings. While DMTs are commonly found in fungi and plants, their presence in bacteria has not been reported. Moreover, the biosynthetic pathways for DMTs have been primarily elucidated in fungi, with identified P450s only acting on the B ring. In this study, we isolated and characterized three bacterial DMTs, namely 3ß-hydroxydrimenol (2), 2α-hydroxydrimenol (3), and 3-ketodrimenol (4), from Streptomyces clavuligerus. Through genome mining and heterologous expression, we identified a cav biosynthetic gene cluster responsible for the biosynthesis of DMTs 2-4, along with a P450, CavA, responsible for introducing the C-2 and C-3 hydroxy groups. Furthermore, the substrate scope of CavA revealed its ability to hydroxylate drimenol analogs. This discovery not only broadens the known chemical diversity of DMTs from bacteria, but also provides new insights into DMT biosynthesis in bacteria.

Deciphering magnesium binding site and structure-function insights in a class II sesquiterpene cyclase.

Magnesium ions (Mg2+) are crucial in class II terpene cyclases that utilize substrates with diphosphate groups. Interestingly, these enzymes catalyze reactions without cleaving the diphosphate group, instead initiating the reaction through protonation. In our recent research, we discovered a novel class II sesquiterpene cyclase in Streptomyces showdoensis. Notably, we determined its crystal structure and identified Mg2+ within its active site. This finding has shed light on the previously elusive question of Mg2+ binding in class II terpene cyclases. In this chapter, we outline our methods for discovering this novel enzyme, including steps for its purification, crystallization, and kinetic analysis.