Mutation of the eunicellane synthase Bnd4 alters its product profile and expands its prenylation ability

Xu, Bingxiang; Ning, Wenbo; Wei, Xiuting; Rudolf, Jeffrey D.

doi:10.1039/d2ob01931k

Cited by 7 publications

(10 citation statements)

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“…In addition, it was reported that the Tpn3 homolog Cyc2 accepts both labdane and clerodane scaffolds 36 , demonstrating the potential substrate promiscuity needed for the investigation of Tpn2 mutants. Second, we utilized our recently reported MKI4 system, a GGPP overproduction system that leverages two promiscuous kinases to phosphorylate exogenously added isoprenol 37,38 , to further enhance diterpene production in vivo (Supplementary Fig. 8).…”

Section: Resultsmentioning

confidence: 99%

Structure-guided product determination of the bacterial type II diterpene synthase Tpn2

et al. 2022

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A grand challenge in terpene synthase (TS) enzymology is the ability to predict function from protein sequence. Given the limited number of characterized bacterial TSs and significant sequence diversities between them and their eukaryotic counterparts, this is currently impossible. To contribute towards understanding the sequence-structure-function relationships of type II bacterial TSs, we determined the structure of the terpentedienyl diphosphate synthase Tpn2 from Kitasatospora sp. CB02891 by X-ray crystallography and made structure-guided mutants to probe its mechanism. Substitution of a glycine into a basic residue changed the product preference from the clerodane skeleton to a syn-labdane skeleton, resulting in the first syn-labdane identified from a bacterial TS. Understanding how a single residue can dictate the cyclization pattern in Tpn2, along with detailed bioinformatics analysis of bacterial type II TSs, sets the stage for the investigation of the functional scope of bacterial type II TSs and the discovery of novel bacterial terpenoids.

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Section: Resultsmentioning

confidence: 99%

Structure-guided product determination of the bacterial type II diterpene synthase Tpn2

et al. 2022

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“…During submission of this work, a remarkable paper on Bnd4 mutagenesis was published 74 . Interestingly, some Bnd4 mutants also generate 7, 13, and 14 as VenA mutants.…”

Section: Discussionmentioning

confidence: 99%

Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase

Jiang

et al. 2023

Nat Commun

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Diterpene synthase VenA is responsible for assembling venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. VenA also demonstrates substrate promiscuity by accepting geranyl pyrophosphate and farnesyl pyrophosphate as alternative substrates. Herein, we report the crystal structures of VenA in both apo form and holo form in complex with a trinuclear magnesium cluster and pyrophosphate group. Functional and structural investigations on the atypical 115DSFVSD120 motif of VenA, versus the canonical Asp-rich motif of DDXX(X)D/E, reveal that the absent second Asp of canonical motif is functionally replaced by Ser116 and Gln83, together with bioinformatics analysis identifying a hidden subclass of type I microbial terpene synthases. Further structural analysis, multiscale computational simulations, and structure-directed mutagenesis provide significant mechanistic insights into the substrate selectivity and catalytic promiscuity of VenA. Finally, VenA is semi-rationally engineered into a sesterterpene synthase to recognize the larger substrate geranylfarnesyl pyrophosphate.

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“…Two other positions, Glu169 and Arg173, also likely have roles in binding Mg 2+ ions or the diphosphate moiety, respectively, as mutation at those positions completely abolished activity. Mutation of some aromatic residues in the active site of Bnd4, particularly W316, altered product formation to include cembrenes (Xu et al., 2022 ). While it is not unusual to see cembrene formation in TS variants (Driller et al., 2019 ) or by simply changing reaction conditions (Rinkel et al., 2018 ), this result was intriguing considering the mechanism of eunicellane formation in coral TSs runs through the cembrenyl cation F + .…”

Section: Mechanisms Of Eunicellane Synthasesmentioning

confidence: 99%

“…If a simple change to Bnd4 adjusts initial cyclization from 10-membered to 14-membering ring formation, continued engineering may be able to divert activity to a coral-like mechanism. Similarly, engineering of cembrene synthases may yield novel eunicellane synthases, although initial attempts with DtCycA were unsuccessful (Xu et al., 2022 ).…”

Section: Mechanisms Of Eunicellane Synthasesmentioning

confidence: 99%

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Biosynthesis, enzymology, and future of eunicellane diterpenoids

Li,

Rudolf

2023

Journal of Industrial Microbiology and Biotechnology

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Eunicellane diterpenoids are a remarkable family of terpene natural products and have been of high interest for over five decades. Widely distributed in soft corals and rare in plants, eunicellanes were also recently identified in actinobacteria. These terpenoids have foundational 6/10-bicyclic frameworks that are frequently oxidized into structures containing transannular ether bridges. Interest in their unique structures and promising biological activities, such as the paclitaxel-like activities of eleutherobin and the sarcodictyins, has led to advancements in natural product isolation, total synthesis, medicinal chemistry, and drug lead development. Until recently, however, there was little known about the biosynthesis and enzymology of these natural products, but several recent studies in both bacteria and coral have opened up the field. This review summarizes recent advancements in the biosynthesis and enzymology of eunicellane diterpenoids and highlights future research prospects in the field. One-Sentence Summary A summary of recent advancements in the biosynthesis and enzymology of eunicellane diterpenoids, a structurally unique and biologically active family of natural products found in coral, plants, and bacteria.

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Mutation of the eunicellane synthase Bnd4 alters its product profile and expands its prenylation ability

Abstract: Bnd4 catalyzes the first committed step in the bio-synthesis of the bacterial diterpenoid benditerpe-noic acid and was the first eunicellane synthase identified from nature. We investigated the catalytic roles of...

Cited by 7 publications

References 28 publications

Structure-guided product determination of the bacterial type II diterpene synthase Tpn2

Structure-guided product determination of the bacterial type II diterpene synthase Tpn2

Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase

Biosynthesis, enzymology, and future of eunicellane diterpenoids

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