A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity: mechanism of biosynthesis of epoxyalcohols

The Journal of Biological Chemistry
Mirela Cristea, Ernst H Oliw

Abstract

Lipoxygenases with R stereospecificity have a conserved Gly residue, whereas (S)-lipoxygenases have an Ala residue. Site-directed mutagenesis has shown that these residues control position and S/R stereospecificity of oxygenation. Recombinant Mn-LO was expressed in Pichia pastoris, and its conserved Gly-316 residue was mutated to Ala, Ser, Val, and Thr. The G316A mutant was catalytically active. We compared the catalytic properties of Mn-LO and the G316A mutant with 17:3n-3, 18:2n-6, 18:3n-3, and 19:3n-3 as substrates. Increasing the fatty acid chain length from C17 to C19 shifted the oxygenation by Mn-LO from the n-6 toward the n-8 carbon. The G316A mutant increased the oxygenation at the n-8 carbon of 17:3n-3 and at the n-10 carbon of the C17 and C18 fatty acids (from 1-2% to 7-11%). The most striking effect of the G316A mutant was a 2-, 7-, and 15-fold increase in transformation of the n-6 hydroperoxides of 19:3n-3, 18:3n-3, and 17:3n-3, respectively, to keto fatty acids and epoxyalcohols. The n-3 double bond was essential. An experiment under an oxygen-18 atmosphere showed that both oxygen atoms were retained in the epoxyalcohols. (R)-Hydroperoxides at n-6 of C17:3, 18:3, and 19:3 were transformed 5 times faster than S ster...Continue Reading

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Citations

Jul 24, 2012·The Journal of Biological Chemistry·Anneli WennmanErnst H Oliw
Oct 24, 2014·Accounts of Chemical Research·Betty J Gaffney
Jun 23, 2009·The New Phytologist·Giuliana D'IppolitoAngelo Fontana
Feb 2, 2011·The FEBS Journal·Florian Brodhun, Ivo Feussner
Nov 28, 2013·Applied Microbiology and Biotechnology·Ruud HeshofLeo H de Graaff
Jan 20, 2016·The Journal of Biological Chemistry·Anneli WennmanYang Chen
Mar 13, 2010·The Journal of Biological Chemistry·Alexandra AndreouIvo Feussner

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