Comment on “Substrate Folding Modes in Trichodiene Synthase: A Determinant of Chemo- and Stereoselectivity”

Abstract

Wang et al. recently reported an in silico study of the trichodiene synthase (TDS) conversion of farnesyl diphosphate (FPP) to trichodiene (TD) (Wang et al., ACS Catal. 2017, 7, 5841–5846). Although the methods and level of theory used in that work are nearly identical to our own recent work on this system (Dixit et al., ACS Catal. 2017, 7, 812–818), Wang et al. reach rather different conclusions. The authors claimed to obtain a “very credible” mechanism for the biosynthesis of TD and optimized the optimal folding mode of FPP in the 1,6-ring closure in TDS. However, the folding mode of the FPP substrate that was presented contradicts well-established NMR and mass spectrometry data. Moreover, the authors make numerous incorrect statements regarding our earlier work.

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

“…The computational protocol adopted here is based on our previous multiscale studies of mono-, sesqui-, and diterpene systems. ,,− , Models of CotB2 with bound ligands were generated using the recently resolved crystal structure . In this crystal structure (PDB code 6GGI, 1.80 Å resolution) 2-fluoro-3,7,18-dolabellatriene, which mimics carbocation A , is bound in the active site, as well as a pyrophosphate moiety and 3 Mg 2+ ions.…”

“…(4) A bifacial active site, where one face is highly charged (composed of charged and polar amino acids, pyrophosphate, bivalent cations, and specific water molecules), while the opposite face is highly hydrophobic and often rich in π-systems . The specific active site topology enables electrostatic reaction guidance and control. ,− (5) Specific positioning of an active site water molecule or acids/bases that allow regioselective substrate (de)­protonation, hydroxylation, or epoxidation. ,, …”

QM/MM study of the taxadiene synthase mechanism

“…S10) yields an energy profile that differs rather strongly from the others and is more in line with the FHM results (see Supporting Information Table S9); we note that the orientation of the cation with respect to PPi differs in the W1E1 and W1E2E snapshots (see Supporting Information Table S10). In general, it is very difficult to predict the correct bound state of the substrate, intermediates, and product in terpene synthases . This is mainly due to the absence of hydrogen bonds between the ligand and the enzyme that could serve as anchors to keep the former in place .…”

Predicting Productive Binding Modes for Substrates and Carbocation Intermediates in Terpene Synthases—Bornyl Diphosphate Synthase As a Representative Case

“…The application of TerDockin specifically to BPPS allowed us to: (1) recapitulate the outcome of a key 18 O labelling experiment, (2) predict an orientation of the product that is consistent with that found in the crystal structure of the BPPS-product complex (and that is similar to that used in previous QM/MM MD simulations), (3) correctly predict the diastereoselectivity for reincorporation of the diphosphate to form the final product, and (4) correctly predict the enantioselectivity of the BPPS reaction. As stated by Major, “the promiscuous binding in terpene synthases is one of the greatest challenges to theory due to weak interactions between the substrate and the hydrophobic pocket in the active site,” 40 The TerDockin method addresses this challenge.…”

“…27–34 These results are in accord with the long-standing hypothesis that one key role of terpene synthases is to pre-organize the substrate such that, once a carbocation is formed in a terpene synthase active site, it quickly reacts to give the known product. 35–40…”