Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase

Proceedings of the National Academy of Sciences of the United States of America
Narayanan SrividyaB Markus Lange

Abstract

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The poten...Continue Reading

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Citations

Feb 5, 2016·Proceedings of the National Academy of Sciences of the United States of America·Patrick SchrepferThomas Brück
Jun 6, 2019·Beilstein Journal of Organic Chemistry·Jan Rinkel, Jeroen S Dickschat
May 20, 2020·Scientific Reports·Ryan W ChristianAmit Dhingra
May 26, 2017·Nature Communications·Tyler P KormanJames U Bowie
Jul 6, 2018·Planta·Cinzia Formighieri, Anastasios Melis
Oct 13, 2018·Journal of Experimental Botany·Glenn W TurnerB Markus Lange
May 13, 2019·Science China. Life Sciences·Qingwen ChenGuodong Wang
Oct 22, 2019·Frontiers in Plant Science·Prema S Karunanithi, Philipp Zerbe
Apr 16, 2020·Biochemistry·Narayanan SrividyaB Markus Lange
Feb 23, 2021·ACS Catalysis·Prabhakar L SrivastavaMarc W van der Kamp
Feb 4, 2019·Phytochemistry·Luani R de O PivaJeffrey Q Chambers
May 5, 2020·ACS Synthetic Biology·Max J KschowakMarkus Buchhaupt
Oct 21, 2021·Chembiochem : a European Journal of Chemical Biology·Nicole G H Leferink, Nigel S Scrutton

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