TRICYCLE

Quantitative exploration of catalytic landscapes underlying the functional divergence of triterpene synthases

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 Obiettivo del progetto (Objective)

'Plants have evolved the ability to produce diverse terpenes to communicate, thrive and survive in various ecologies and these have profound benefits for humanity as therapeutics and bioactive compounds. While chemical novelty has contributed to the success of sessile organisms like plants, the evolutionary origins of terepene complexity are poorly understood at the molecular level. Among terpenes, triterpenes are produced by oxidosqualene cyclase (OSC’s) family members. In OSC family, beta-amyrin synthases (BAS) is involved in triterpene secondary metabolism and functionally diverged from but is closely related to cycloartenol synthases (CAS) of sterol primary metabolism. Structural changes that interconnect this pair of enzymes remain unknown and hence form a prime model system to address many basic questions; what are/were the key structural changes responsible for the conversion of CAS to BAS?Is this transition unique or a recurring theme? How has this functional transition occurred in other CAS contexts?To approach these questions, project quantitatively explores catalytic landscape subtended by extant CA and BA synthases of plants. Catalytic landscaping is a novel concept that uses multidisciplinary approaches to discern the structural changes that interconnect a pair of recently diverging enzymes. Elucidating the catalytic landscape of CAS and BAS would yield important insights into the evolution of triterpene complexity as a whole and aid applied efforts to engineer pathways for novel triterpenes. Project aims to; i)computational and structural modelling of CAS and BAS to identify functionally relevant amino acids to enable ii)in silico design and synthesis of mutant library in CA background using structure-based combinatorial protein engineering. Library will be iii) expressed in yeast to identify and quantify triterpenes using GC-MS. iv)analysis of data to build models to relate changes in product specificity to the underlying structural changes in CAS-BAS.'

Introduzione (Teaser)

Researchers have re-engineered a common plant protein to produce various novel compounds within the terpenes group.

Descrizione progetto (Article)

Terpenes are a broad class of plant-produced compounds commercially valuable as health-modifying compounds, building blocks for more complex molecules and essential oils. They can by synthesised using the oxidosqualene cyclase (OSC) enzyme family.

The EU-funded TRICYCLE project aimed to understand how changing the structures of OSC enzymes could alter the terpene compounds they produce. They used a technique called catalytic landscaping to achieve this.

Researchers studied a large number of OSC enzymes to identify which amino acid residues (the building blocks of proteins like these enzymes) were involved in the catalysis of terpenes. This analysis revealed eight amino acids that controlled which terpenes were made by the OSC enzymes.

Using this information, TRICYCLE created a number of mutated enzymes by changing these amino acids. The mutated enzymes were expressed in yeast, and researchers studied the resulting terpene products.

They found that just a few mutations in the OSC enzymes resulted in major changes in the types of terpenes produced. Additionally, several of the altered enzymes produced never-before-seen terpenes.

This research could prove very valuable in the search for potential new drugs. In addition, it has improved our understanding of terpenes, an important class of plant-produced compounds.