Mutational analysis of LinD suggests transformations are catalyzed using non-covalent mechanisms

The interconversion of non-activated alkenes and alcohols, catalysed by (de)hydratases, has great potential in biotechnology for the generation of fine and bulk chemicals. LinD is a cofactor-independent enzyme that catalyses the reversible (de)hydration of the tertiary alcohol (S)-linalool to the triene b-myrcene, and also its isomerization to the primary alcohol geraniol.

In this collaboration project between Dr. A. Cuetos and Prof. G. Grogan from the University of York (UK) and Dr. J. Iglesias-Fernández, and Prof. S. Osuna from IQCC, structure-informed mutagenesis of LinD, followed by activity and mechanistic studies were performed. The combined experimental-computational approach confirmed essential roles for residues C171, C180 and H129 in water activation for the hydration of b-myrcene to linalool. No evidence of covalent thioterpene intermediates was found using either X-ray crystallography, or mass spectrometry, which was further confirmed by means of QM/MM nudged elastic band simulations. Labelling and NMR experiments confirmed a role for residue D39 in (de)protonation of the linalool carbon C10 in the isomerization of linalool to geraniol and also the intermediacy of b-myrcene in this isomerization reaction.  X-ray, molecular dynamics and activity studies also suggested a significant role in catalysis for a mobile methionine residue M125, which exists in substantially altered orientations in different mutant structures.

The paper was published recently in ACS Catalysis:

A. Cuetos, J. Iglesias-Fernandez, H. Danesh-Azari, E. Zukic, A. Dowle, S. Osuna, and G. Grogan
“Mutational Analysis of Linalool Dehydratase-Isomerase (LinD) Suggests Alcohol and Alkene Transformations are Catalyzed Using Non-Covalent Mechanisms”
ACS Catal. 2020, ASAP
DOI: 10.1021/acscatal.0c02958

Girona, September 12, 2020
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