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Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase

Chen, Mengbin, Chou, Wayne K. W., Al-Lami, Naeemah, Faraldos, Juan, Allemann, Rudolf, Cane, David E. and Christianson, David W. 2016. Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase. Biochemistry 55 (20) , pp. 2864-2874. 10.1021/acs.biochem.6b00343

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Abstract

Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously-determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule is coordinated to the Mg2+C ion. There is no obvious role for water in the ATAS mechanism, since the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg2+C-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity of Mg2+C-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis, but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Chemistry
Publisher: ACS Publications
ISSN: 00062960
Funders: BBSRC
Date of First Compliant Deposit: 10 August 2016
Date of Acceptance: 12 May 2016
Last Modified: 04 Jun 2017 09:21
URI: http://orca.cf.ac.uk/id/eprint/93708

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