Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

Abstract

Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.computational biology | closed complex modeling | protein engineering | terpene synthases | terpene synthase catalysis

Molecular dynamics study of taxadiene synthase catalysis

“…This transformation is the first committed step in the biosynthesis of taxol, a natural tetracyclic diterpene (originally isolated from the pacific yew tree Taxus brevifolia ), which has potent anticancer activity and is clinically applied in the first line treatment of breast, lung, and ovarian cancer . Understanding the molecular basis of TXS catalysis constitutes an active field of research, as it may enable sustainable biotechnological pathways for taxol production that could substitute conventional semi‐synthetic routes, which are associated with significant costs and the accumulation of significant toxic waste streams …”

“…In red: minor products of TXS catalysis. Product yields (numbers in blue) are given as reported by Schrepfer et al The numbering of GGPP is used for the carbocation intermediates, while minor products are numbered according to taxadiene convention. OPP denotes the diphosphate group of GGPP.…”

“… Representative structure of TXS in the closed active site conformation. Atomic coordinates were provided by Schrepfer et al The protein regions capping the active site are shown in different colors: loops A–C in blue, J–K in cyan, and H–α1 in purple. The N terminus random coil (NTRC) segment is shown in orange, while the C‐terminal portion of helix H is shown in yellow like the rest of the protein.…”

“…Several experimental and computational studies have been reported which provide insights into TXS catalysis . However, the catalytic role of the enzyme along the entire reaction path (from GGPP to T ) is not yet defined in detail.…”

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

“…Recently reported targeted engineering [51] of the Class I taxadiene synthase from Taxus brevifolia (TXS) enabled new understanding of the mechanistic procedures that are carried out by this enzyme on the substrate GGPP. Quenching the carbocation cascade that naturally leads to the formation of tricyclic taxadiene [56] was achieved by exchanging a valin in position 584 with methionine.…”