Identification of amino acid networks governing PNAS PLUS catalysis in the closed complex of class I terpene synthases Patrick Schrepfera, Alexander Buettnera, Christian Goernera, Michael Hertela, Jeaphianne van Rijnb, Frank Wallrappc, Wolfgang Eisenreichd, Volker Siebere, Robert Kouristf, and Thomas Brücka,1 aDivision for Industrial Biocatalysis, Technische Universität München, 85748 Garching, Germany; bDepartment of Theoretical Chemistry, Max-Planck-Institut für Kohlenforschung, 45470 Mülheim an der Ruhr, Germany; cChair of Bioinformatics and Computational Biology, Technische Universität München, 85748 Garching, Germany; dChair of Biochemistry, Technische Universität München, 85748 Garching, Germany; eChair of Chemistry of Biogenic Resources, Technische Universität München, 94315 Straubing, Germany; and fJunior Research Group for Microbial Biotechnology, Ruhr-Universität Bochum, 44780 Bochum, Germany Edited by David H. Sherman, University of Michigan, Ann Arbor, MI, and accepted by the Editorial Board January 5, 2016 (received for review October 4, 2015) Class I terpene synthases generate the structural core of bioactive nistic considerations explaining this promiscuity on the molecular terpenoids. Deciphering structure–function relationships in the reac- level are very limited (8, 21–25). The role of the protein scaffold has tive closed complex and targeted engineering is hampered by highly been widely thought to chaperone the cyclization cascade by merely dynamic carbocation rearrangements during catalysis. Available crys- conformational control (22, 26). Very recently, a hypothesis point- tal structures, however, represent the open, catalytically inactive ing to a possible involvement of electrostatic effects during the form or harbor nonproductive substrate analogs. Here, we present carbocation cascade of other terpene synthases has been proposed. a catalytically relevant, closed conformation of taxadiene synthase These effects are thought to be mediated by the PP anion co- (TXS), the model class I terpene synthase, which simulates the initial i product that may be retained in the active site (21, 24, 26–32). catalytic time point. In silico modeling of subsequent catalytic steps A crystal structure of the open, catalytically inactive TXS allowed unprecedented insights into the dynamic reaction cascades conformation containing an unproductive fluorinated substrate and promiscuity mechanisms of class I terpene synthases. This gen- BIOPHYSICS AND analog (2-F-GGPP) in the active site is available [Protein Data erally applicable methodology enables the active-site localization of COMPUTATIONAL BIOLOGY carbocations and demonstrates the presence of an active-site base Bank (PDB) ID code 3P5R] (8, 15). This dataset, however, only motif and its dominating role during catalysis. It additionally allowed provides limited information on the dynamic carbocation pro- in silico-designed targeted protein engineering that unlocked the cesses involved in the cyclization cascade. At present, none of the path to alternate monocyclic and bicyclic synthons representing the reported mechanistic or structural studies on class I terpene basis of a myriad of bioactive terpenoids. synthases do consider detailed structure–function relationships of the catalytically active enzyme conformation. This situation computational biology | closed complex modeling | protein engineering | arises as the use of substrate analogs and crystal structures of terpene synthases | terpene synthase catalysis open conformations do not reflect the concerted, dynamic events inside the closed, catalytically active enzyme complex. In fact, the erpene synthases transform aliphatic allylic diphosphate closed enzyme complex containing a native GGPP substrate has Tprecursors to complex macrocycles, which represent the structural core of numerous bioactive terpenoids (1). Despite Significance their low primary sequence identity, terpene synthases share highly conserved tertiary and quaternary structural features, Class I terpene synthases are essential in biosynthesis of bio- α which are dominated by -helical barrel folds (1, 2). Taxadiene active terpenoids (e.g., Taxol). Identification of structure– Taxus brevifolia synthase (TXS/ ) catalyzes the cyclization of the function correlations is hampered by highly dynamic carbocation- E E E universal diterpene precursor ( , , )-geranylgeranyl diphosphate driven reactions and the limited availability of catalytically (GGPP, C20) to taxa-4,11-diene (taxadiene, T) (Fig. 1), the first relevant crystal structures. We provide the closed, catalytically committed biosynthetic step toward the clinically important tumor active conformations of taxadiene synthase (TXS) and various therapeutic Taxol (3–6). TXS, a class I diterpene synthase, uses a taxonomically unrelated class I terpene synthases in complex 2+ trinuclear Mg ion cluster coordinated by two conserved binding with their catalytically relevant carbocationic intermediates. motifs [DDXXD and (N,D)DXX(S,T)XXXE] to initiate catalysis. Our methodology allows direct prediction and validation of 2+ The Mg ion cluster facilitates orientation of the GGPP pyro- universal structural features that govern the highly dynamic phosphate moiety (PPi) in the active site, followed by active-site catalytic processes of class I terpene synthases. Our data en- closure and GGPP ionization (7, 8). Although native GGPP-derived abled delineation of a discrete reaction pathway for TXS and carbocation intermediates could hitherto not be trapped experi- allowed targeted enzyme engineering to generate alter- mentally, mechanistic studies using GGPP analogs and quantum nate macrocyclic core structures, which can act as synthons for chemical calculations [quantum mechanics (QM)] allowed de- bioactive lead structures. lineation of several potential carbocation intermediates (Fig. 1) (9–15). However, the nature of the deprotonating base directing Author contributions: P.S. and T.B. conceived the study; P.S., F.W., W.E., V.S., R.K., and T.B. designed the experimental setup; P.S., A.B., M.H., and R.K. performed research; formation of T remains elusive. Monocyclic and bicyclic carbocations P.S., C.G., J.v.R., F.W., W.E., and R.K. analyzed data; P.S., W.E., V.S., R.K., and T.B. wrote postulated to form during taxadiene formation, structurally re- the paper. – semble the macrocyclic core of other bioactive diterpenoids (16 The authors declare no conflict of interest. 20). The molecular mechanism of TXS, which can be considered This article is a PNAS Direct Submission. D.H.S. is a guest editor invited by the Editorial as template for all class I terpene synthases, may therefore improve Board. a general understanding of terpene synthase mechanisms. TXS and 1To whom correspondence should be addressed. Email: [email protected]. other class I terpene synthases produce side-products [e.g., taxa- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 4(20),11-diene, T1] (Fig. 1). However, structural data and mecha- 1073/pnas.1519680113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1519680113 PNAS Early Edition | 1of10 Downloaded by guest on September 28, 2021 Fig. 1. Cascade pathway, carbocation rearrangements, and deprotonated intermediates. The black box depicts the postulated carbocationic intermediates during the cascade pathway from GGPP→T and the numbering of the carbocation cascade steps. The red boxes depict the found deprotonated intermediates, whereby the red arrows indicate the cationic intermediate from which the deprotonated intermediate is derived. The structure in the middle depicts the + conformation of productive GGPP (blue, double bonds in yellow) and the localization of the trinuclear Mg2 cluster (green) in the active site of the closed TXS conformation. Note that numbering of cationic and deprotonated intermediates differs from previous reports (9, 11, 12, 15). not been considered to decipher native structure–function cor- Results relations and catalytic mechanisms of terpene synthases. TXS•GGPP Complex: Evolutionarily Conserved Motifs Initiate the In this study, we constructed a closed, active TXS conforma- Carbocation Cascade. The reported crystal structure of TXS, tion harboring native GGPP in its productive conformation. To representing the open, catalytically inactive enzyme, contains elucidate the catalytically relevant complexes, reflecting the 2-F-GGPP in an unproductive orientation leading to subsequent carbocation cascade in the active site as well as structure–func- intermediates in which several stereocenters are inverted (Fig. tion relationships, we further applied a molecular mechanic 2A) (15). Using molecular mechanics, we constructed a model of (MM)-based modeling approach combined with site-directed theclosedTXS•GGPP complex. This complex is considered to mutagenesis-guided experimental verification. The data provide be the catalytically active conformation of the enzyme and rep- new, unprecedented insights into the control of the cyclization resents the initial step of the catalytic carbocation cascade (Fig. mechanism and the product promiscuity of class I terpene syn- 2B and SI Appendix,Fig.S1A). The model omits the 80-residue thases. Application of in silico-guided protein engineering enabled N-terminal transit sequence. To provide for correct substrate selective attenuation of the carbocation cascade at several in- folding, we manually docked a geometry-optimized GGPP struc- termediate steps. The methodology allowed experimental confir- ture, derived