The intricate mechanisms in the biosynthesis of terpenes belong to the most challenging problems in natural product chemistry. Methods to address these problems include the structure-based site-directed mutagenesis of terpene synthases, computational approaches, and isotopic labeling experiments. The latter approach has a long tradition in biosynthesis studies and has recently experienced a revival, after genome sequencing enabled rapid access to biosynthetic genes and enzymes. Today, this allows for a combined approach in which isotopically labeled substrates can be incubated with recombinant terpene synthases. These clearly defined reaction setups can give detailed mechanistic insights into the reactions catalyzed by terpene synthases, and recent developments have substantially deepened our understanding of terpene biosynthesis. This chapter will discuss the state of the art and introduce some of the most important methods that make use of isotopic labelings in mechanistic studies on terpene synthases.

Proanthocyanidins (PAs) are natural flavan-3-ol polymers that contribute protection to plants under biotic and abiotic stress, benefits to human health, and bitterness and astringency to food products. They are also potential targets for carbon sequestration for climate mitigation. In recent years, from model species to commercial crops, research has moved closer to elucidating the flux control and channeling, subunit biosynthesis and polymerization, transport mechanisms, and regulatory networks involved in plant PA metabolism. This review extends the conventional understanding with recent findings that provide new insights to address lingering questions and focus strategies for manipulating PA traits in plants.

A newly identified bacterial (Z)-γ-bisabolene synthase was used for investigating the cyclisation mechanism of the sesquiterpene. Since the stereoinformation of both chiral putative intermediates, nerolidyl diphosphate (NPP) and the bisabolyl cation, is lost during formation of the achiral product, the intriguing question of their absolute configurations was addressed by incubating both enantiomers of NPP with the recombinant enzyme, which resolved in an exclusive cyclisation of (R)-NPP, while (S)-NPP that is non-natural to the (Z)-γ-bisabolene synthase was specifically converted into (E)-β-farnesene. A hypothetical enzyme mechanistic model that explains these observations is presented.