QM/MM study of the taxadiene synthase mechanism

Abstract

Combined quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the reaction mechanism of taxadiene synthase (TXS). TXS catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to taxadiene (T) and four minor cyclic products. All these products originate from the deprotonation of carbocation intermediates. The reaction profiles for the conversion of GGPP to T as well as to minor products were calculated for different configurations of relevant TXS carbocation complexes. The QM region was treated at the M06-2X/TZVP level, while the CHARMM27 force field was used to describe the MM region. The QM/MM calculations suggest a reaction pathway for the conversion of GGPP to T, which slightly differs from previous proposals regarding the number of reaction steps and the conformation of the carbocations. The QM/MM results also indicate that the formation of minor products via water-assisted deprotonation of the carbocations is highly exothermic, by about −7 to −23 kcal/mol. Curiously, however, the computed barriers and reaction energies indicate that the formation of some of the minor products is more facile than the formation of T. Thus, the present QM/MM calculations provide detailed insights into possible reaction pathways and into the origin of the promiscuity of TXS, but they do not reproduce the product distribution observed experimentally.

Enhanced production of taxadiene in Saccharomyces cerevisiae

“…The mass spectrum of diterpene 1 closely resembled cembrene-type diterpene (cembrene A) produced by neocembrene synthase in S. cerevisiae [ 30 ]. The cyclisation of GGPP to taxadiene involves production of a cembrenyl intermediate [ 31 ]. The relatively high degree of similarity suggests the compound observed in this study could potentially be a side product of this cyclisation step.…”

Interrogating chemical mechanisms in natural products biosynthesis using quantum chemical calculations

“…While these differences leave many mechanistic issues unresolved, they highlight the sensitivity of results to both differences in procedural details and differences in enzyme conformation and they point to the need for new homology modeling and docking methods, perhaps specific to terpene synthases. [57][58][59][60] As pointed out by Thiel and coworkers, 56 this situation may not represent a failure in modeling but, instead, the different models may correspond to different biologically relevant states of taxadiene synthase. It will take clever (bio)chemists to propose new experimental tests of the validity of each model and distinguish between them.…”

“…The results of enzyme free F I G U R E 7 Bifurcating pathway en route to abietadiene F I G U R E 8 Portion of the taxadiene forming carbocation cascade computations at a variety of different levels indicate that both direct and two-step proton transfer processes are energetically viable, although the former is energetically preferred. [49][50][51][52][53][54][55][56] Brück and coworkers docked optimized carbocations into taxadiene synthase, 52 while QM/MM MD simulations were carried out by the Major 53,54 and Thiel 55,56 groups using DFT methods for the QM region (M06-2X/6-31+G(d,p) and SCC-DFTB, respectively). Given the lack of a complete X-ray crystal structure of taxadiene synthase in a catalytically competent closed form, homology models were generated before incorporation of carbocations; the approaches used and resulting structures differed from group to group.…”

Biosynthesis of paclitaxel using synthetic biology

“…to remove the PPi group and forms a carbocation at GGPP. After an array of rearrangements, GGPP is converted into taxa-4(5),11(12)-diene (taxadiene, 93.2 %), taxa-4(20),11(12)-diene (iso-taxadiene, 4.7%) and other byproducts (less than 3%) (van Rijn et al 2019). Moreover, TXS contains protein folds characteristic of both class I and class II cyclases, but has catalytic activity only in the class I domain (Ko ¨ksal et al 2011;Zhou et al 2012).…”