Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(7)

Published: Feb. 6, 2024

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, their biosynthetic construction has been subject intensive study for many years. The enzymatic basis the production aspidosperma iboga alkaloids, which produced exclusively by members Apocynaceae family, recently discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus Tabernanthe catalyze regio- enantiodivergent [4+2] cycloaddition reactions to generate (tabersonine synthase, TS) (coronaridine CorS; catharanthine CS) scaffolds common intermediate. Here, we use combined phylogenetic biochemical approach investigate evolution functional diversification these cyclase enzymes. Through ancestral sequence reconstruction, provide evidence initial TS an CXE followed emergence CorS in two separate lineages, leading turn CS genus. This progression alkaloid biosynthesis is consistent with chemotaxonomic distribution MIAs. We subsequently test panel chimeras based on cyclases probe molecular differential cyclization activity. Finally, show through partial heterologous reconstitution tabersonine using non-pathway how could have first appeared as “underground metabolites” via recruitment promiscuous protein families. Our results insight into new secondary metabolic pathways can emerge small but important changes following co-option preexisting functions.

Language: Английский

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Discovery and evolution of [4 + 2] cyclases

Current Opinion in Chemical Biology, Journal Year: 2024, Volume and Issue: 81, P. 102504 - 102504

Published: July 27, 2024

Language: Английский

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Genome‐based discovery of pachysiphine synthases in Tabernaemontana elegans

The Plant Journal, Journal Year: 2024, Volume and Issue: 120(5), P. 1880 - 1900

Published: Oct. 20, 2024

Plant-specialized metabolism represents an inexhaustible source of active molecules, some which have been used in human health for decades. Among these, monoterpene indole alkaloids (MIAs) include a wide range valuable compounds with anticancer, antihypertensive, or neuroactive properties. This is particularly the case pachysiphine derivatives show interesting antitumor and anti-Alzheimer activities but accumulate at very low levels several Tabernaemontana species. Unfortunately, genome data Tabernaemontanaceae are lacking knowledge on biogenesis pachysiphine-related MIAs planta remains scarce, limiting prospects biotechnological supply many pachysiphine-derived biopharmaceuticals. Here, we report raw version toad tree (Tabernaemontana elegans) sequence. These new genomic resources led to identification characterization couple genes encoding cytochrome P450 synthase activity. Our phylogenomic docking analyses highlight different evolutionary processes that recruited epoxidize precursor tabersonine specific position dedicated orientation, thus enriching our understanding diversification speciation MIA plants. gene discoveries also allowed us engineer synthesis yeast through combinatorial association metabolic enzymes resulting tailor-made non-natural MIAs. Overall, this work step forward future drugs by microbial cell factories.

Language: Английский

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Evolutionary insights into the stereoselectivity of imine reductases based on ancestral sequence reconstruction

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Nov. 28, 2024

The stereoselectivity of enzymes plays a central role in asymmetric biocatalytic reactions, but there remains dearth evolution-driven biochemistry studies investigating the evolutionary trajectory this vital property. Imine reductases (IREDs) are one such enzyme that possesses excellent stereoselectivity, and stereocomplementary members pervasive family. However, regulatory mechanism behind stereocomplementarity cryptic. Herein, we reconstruct panel active ancestral IREDs trace evolution from ancestors to extant IREDs. Combined with coevolution analysis, reveal six historical mutations capable recapitulating evolution. An investigation X-ray crystallography shows they collectively reshape substrate-binding pocket regulate inversion. In addition, construct an empirical fitness landscape discover epistasis is prevalent Our findings emphasize power ASR circumventing time-consuming large-scale mutagenesis library screening for identifying change functions support Darwinian premise molecular perspective biological stepwise process. There paucity on perspective. Here, authors use sequence reconstruction imine reductases, elucidate its structural basis, epistasis.

Language: Английский

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Chemical tools for unpicking plant specialised metabolic pathways

Current Opinion in Plant Biology, Journal Year: 2024, Volume and Issue: 80, P. 102554 - 102554

Published: May 30, 2024

Elucidating the biochemical pathways of specialised metabolites in plants is key to enable or improve their sustainable biotechnological production. Chemical tools can greatly facilitate discovery biosynthetic genes and enzymes. Here, we summarise transdisciplinary approaches where methods from chemistry chemical biology helped overcome challenges pathway elucidation. Based on recent examples, describe how state-of-the-art isotope labelling experiments guide selection gene candidates, affinity-based probes identification novel enzymes, semisynthesis availability elusive intermediates, biomimetic reactions provide a better understanding inherent reactivity. We anticipate that wider application such will accelerate pace elucidation plants.

Language: Английский

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Evolutionary divergence of CXE gene family in green plants unveils that PtoCXEs overexpression reduces fungal colonization in transgenic Populus

Tree Physiology, Journal Year: 2024, Volume and Issue: 44(7)

Published: June 21, 2024

Abstract Plant enzymes significantly contribute to the rapidly diversified metabolic repertoire since colonization of land by plants. Carboxylesterase is just one ubiquitous, multifunctional and ancient that has particularly during plant evolution. This study provided a status on carboxylesterase landscape within Viridiplantae. A total 784 carboxylesterases were identified from genome 31 species representing nine major lineages sequenced Viridiplantae divided into five clades based phylogenetic analysis. Clade I genes may be bacterial origin then expanded II was first gained in ancestor bryophytes after plants, III IV ferns which considered most advanced seedless vascular while V seed To date, functions woody plants remain unclear. In this study, 51 Populus trichocarpa further eight classes. Tandem segmental duplication events both contributed expansion Populus. Although proven enhance resistance pathogens many herbaceous species, relevant researches forest trees are still needed. pathogen incubation assays showed overexpressing six Class VI tomentosa, greater or lesser degree, reduced detached leaves fungus Cytospora chrysosperma. significant difference also found functional divergence patterns for derived different gene events. Functional differentiation duplicated proved time vivo physiological The identification potentially anti-fungal PtoCXE06 laid theoretical foundation promoting genetic improvement disease-resistance traits trees.

Language: Английский

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Applications of ancestral sequence reconstruction for understanding the evolution of plant specialized metabolism

Philosophical Transactions of the Royal Society B Biological Sciences, Journal Year: 2024, Volume and Issue: 379(1914)

Published: Sept. 29, 2024

Studies of enzymes in modern-day plants have documented the diversity metabolic activities retained by species today but only provide limited insight into how those properties evolved. Ancestral sequence reconstruction (ASR) is an approach that provides statistical estimates ancient plant enzyme sequences which can then be resurrected to test hypotheses about evolution catalytic and pathway assembly. Here, I review insights been obtained using ASR study metabolism highlight important methodological aspects. Overall, studies show (i) exaptation widespread such even low or undetectable levels ancestral activity with a substrate later become apparent primary descendant enzymes, (ii) intramolecular epistasis may not limit evolutionary paths towards preference switches, (iii) flux often differs from networks. These other gained would possible sequences. Future characterizing entire networks as well link structures enzymatic should continue novel chemical This article part theme issue ‘The metabolism’.

Language: Английский

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Genome-based discovery of pachysiphine synthases inTabernaemontana elegans

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 30, 2024

Abstract Plant specialized metabolism represents an inexhaustible source of active molecules, some which have been used in human health for decades. Among these, monoterpene indole alkaloids (MIAs) include a wide range valuable compounds with anticancer, antihypertensive, or neuroactive properties. This is particularly the case pachysiphine derivatives show interesting antitumor and anti-alzheimer activities but accumulate at very low levels several Tabernaemontana species. Unfortunately, genome data Tabernaemontanaceae are lacking knowledge on biogenesis pachysiphine-related MIAs planta remains scarce, limiting prospects biotechnological supply many pachysiphine-derived biopharmaceuticals. Here, we report raw version toad tree ( elegans ) sequence. These new genomic resources led to identification characterization couple genes encoding cytochrome P450 synthase activity. Our phylogenomic docking analyses highlights different evolutionary processes that recruited epoxidize precursor tabersonine specific position dedicated orientation, thus enriching our understanding diversification speciation MIA plants. gene discoveries also allowed us engineer synthesis yeast through combinatorial association metabolic enzymes resulting tailor-made non-natural MIAs. Overall, this work step forward future drugs by microbial cell factories. Significance Statement While alkaloid high interest anti-Alzheimer compound, its biosynthesis involving epoxidation uncharacterized. By sequencing assembling , identified two P450s exhibiting activity modelized explore scenario leading acquisition expoxidase activity; factories securing producing new-to-nature alkaloids.

Language: Английский

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Ancestral Sequence Reconstruction to Enable Biocatalytic Synthesis of Azaphilones

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(44), P. 30194 - 30203

Published: Oct. 23, 2024

Biocatalysis can be powerful in organic synthesis but is often limited by enzymes' substrate scope and selectivity. Developing a biocatalytic step involves identifying an initial enzyme for the target reaction followed optimization through rational design, directed evolution, or both. These steps are time consuming, resource-intensive, require expertise beyond typical chemistry. Thus, effective strategy streamlining process from identification to implementation essential expanding biocatalysis. Here, we present combining bioinformatics-guided mining ancestral sequence reconstruction (ASR) resurrect enzymes synthesis. Specifically, achieve enantioselective of azaphilone natural products using two enzymes: flavin-dependent monooxygenase (FDMO) stereodivergent oxidative dearomatization substrate-selective acyltransferase (AT) acylation enzymatically installed hydroxyl group. This cascade, stereocomplementary established chemoenzymatic routes, expands access enantiomeric linear tricyclic azaphilones. By leveraging co-occurrence coevolution FDMO AT biosynthetic pathways, identified candidate, CazE, addressed its low solubility stability ASR, obtaining more soluble, stable, promiscuous, reactive (AncAT). Sequence analysis revealed AncAT as chimeric composition descendants with enhanced reactivity likely due promiscuity. Flexible receptor docking molecular dynamics simulations showed that most promotes geometry between substrates. We anticipate our bioinformatics-guided, ASR-based approach broadly applied target-oriented synthesis, reducing required develop efficiently superior biocatalysts.

Language: Английский

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Estrogenic actions of alkaloids: Structural characteristics and molecular mechanisms

Biochemical Pharmacology, Journal Year: 2024, Volume and Issue: unknown, P. 116645 - 116645

Published: Nov. 1, 2024

Language: Английский

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