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Flowering plants use a pseudoenzyme to form indole

Plants produce an astonishing variety of chemical substances. Most of these metabolites are involved in chemical communication and serve to defend against pests, protect against pathogens, and attract beneficial organisms. One example is indole, a nitrogen-containing aromatic compound that acts as a central intermediate in the biosynthesis of the amino acid tryptophan.

In many plants, indole also serves as a precursor for defensive substances. Many plants release volatile indole when attacked by herbivores to deter them or warn neighboring plants of an impending attack. This strengthens the plant's resistance. Indole can also attract pollinators as a component of flower scents.

Matilde Florean and Tobias Köllner, who heads a research team in the Department of Natural Product Biosynthesis at the Max Planck Institute for Chemical Ecology, are investigating enzymes involved in the biosynthesis of benzoxazinoids. Benzoxazinoids are specialized defense compounds in plants that require indole as a precursor for production. Their study is in Nature Chemical Biology.

Some plant species, such as maize and larkspur (monocotyledons and basal dicotyledons), have enzymes that are active and responsible for indole biosynthesis. However, in other flowering plants, the core eudicots, these enzymes are only weakly active.

"We therefore suspected that these plants have a different, more efficient mechanism for indole biosynthesis," says Florean, the study's first author.

Pseudoenzyme plays a key role in the formation of indole

The team tested many different enzymes that could be involved in the crucial indole synthesis step using the model plant Nicotiana benthamiana. They also performed enzymatic tests with purified proteins in vitro. Through these experiments, the researchers discovered that indole biosynthesis in these plants is mediated by the pseudoenzyme TSB-like. Pseudoenzymes are proteins that closely resemble active enzymes, yet lack the ability to catalyze reactions.

The pseudoenzyme TSB-like is structurally similar to the enzyme TSB (tryptophan synthase beta subunit); however, unlike TSB, TSB-like is not involved in tryptophan formation. TSB normally binds to and activates another enzyme, TSA (tryptophan synthase alpha subunit). This leads to the brief formation of indole, which is then immediately converted to tryptophan by TSB. TSB-like binds to and activates TSA, but the interaction between TSB-like and TSA does not lead to tryptophan formation. Therefore, indole can be released as a volatile compound.

"Normally we look for enzymes that show catalytic activity for the reaction we are interested in. Those that do not are usually of little interest to us. Therefore, it was surprising to see that indole biosynthesis is mediated by a pseudoenzyme that diverts the activity of an active enzyme to produce indole for the emission of volatiles or the biosynthesis of specialized defense compounds." says Köllner.

Pseudoenzymes are difficult to detect because they lack enzymatic activity without a suitable binding partner. However, they are not rare and can account for up to 10% of the proteome, or the totality of all proteins. In this relatively new research field, scientists still have many questions about these proteins.

The research team now wants to find out how plants regulate the activity of pseudoenzymes such as TSB-like. They are particularly interested in how plants prevent TSB-like from competing with the catalytically active TSB enzyme for the essential tryptophan biosynthesis.

"Unraveling the mechanism by which the large and economically important group of core eudicots produces indole is also relevant for modern agriculture," says Sarah O'Connor, head of the Department of Natural Product Biosynthesis. One possibility is the targeted breeding of plants that are more attractive to pollinators and less susceptible to pathogens and pests.