Computational investigation of the reaction mechanism of aristolochene synthase

Young, Neil James 2009. Computational investigation of the reaction mechanism of aristolochene synthase. PhD Thesis, Cardiff University.

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Abstract

Gas phase ab initio, semi-empirical and density functional theory (DFT) quantum mechanical (QM) calculations are used to investigate key steps in the conversion of farnesyl diphosphate to aristolochene by the sesquiterpene synthase, aristolochene synthase (AS-PR), from P. roquefort Molecular docking and a combined quantum mechanics / molecular dynamics (QM/MM) are then used to simulate the reaction in silico. A total of three models of AS-PR are considered, one with two magnesium ions docked and two models containing three magnesiums ions docked in different positions. Gas phase results indicate that an intramolecular proton transfer, either directly or via a water molecule, during a key step in the mechanism is feasible with a maximum barrier height of ca. 22 kcal mol-1. Such a mechanism would avoid the generation, quenching and regeneration of a high energy carbocation intermediate. However, experimental evidence obtained contemporaneously with these results suggest that AS does indeed utilise an alternative mechanism involving the formation of a neutral intermediate (germacrene A) but the identity of the acid capable of protonating this intermediate has yet to be determined. Molecular docking experiments performed in this work reject a previous suggestion that diphosphate ion can perform this role. A series of QM/MM free energy sampling simulations demonstrate no significant lowering of the energetic barrier to intramolecular proton transfer over the gas phase suggesting that AS-PR does not catalyse this step of the reaction mechanism in vivo. Additional data also seemingly rule out Lys 206 as a possible active site acid. The condensed phase data also support gas phase results which suggest the formation of intermediate eudesmane cation occurs via a concerted process but that subsequent steps axe not concerted and that there are further true on-path intermediates in the reaction mechanism catalysed by AS-PR. By considering a number of different models for the AS-PR holoenzyme the role of the magnesium in causing favourable substrate binding and acting as a 'trigger' for the cyclisation cascade are presented and computational results support the tentative evidence obtained by X-ray crystallography. The results presented in this work add weight to the developing theories in sesquiterpene chemistry in which the biosynthesis of these molecules is achieved by enzymes whose role is the act as chaperones guiding their universal substrate into appropriate conformation, triggering catalysis by the binding of magnesium and preventing premature quenching of reactive carbocations by solvent.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
ISBN:	9781303191343
Date of First Compliant Deposit:	30 March 2016
Last Modified:	10 Jan 2018 05:55
URI:	https://orca.cardiff.ac.uk/id/eprint/55897

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