On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Gianguido Coffa,Ann N Imber,Brendan C Maguire,Gurunathan Laxmikanthan,Claus Schneider,Betty J Gaffney,Alan R Brash

doi:10.1074/jbc.m504870200

Gianguido Coffa, Ann N Imber + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m504870200

Copy DOI

Abstract

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

Highlights

Polyunsaturated fatty acids are oxygenated to signaling molecules of very specific structure by the dioxygenases lipoxygenase (LOX)4 and cyclooxygenase, and by the cytochrome P450 class of monooxygenase [1,2,3]
In both classes of dioxygenase the fatty acid substrate is activated to a radical species capable of instantaneous reaction with molecular oxygen [1, 2]; all elements of reaction specificity depend on arranging the meeting of molecular oxygen with the correct site on the substrate radical
The results of our study are relevant to the issues of substrate access, substrate orientation, and control of oxygenation in soybean LOX-1

Summary

Introduction

Polyunsaturated fatty acids are oxygenated to signaling molecules of very specific structure by the dioxygenases lipoxygenase (LOX) and cyclooxygenase, and by the cytochrome P450 class of monooxygenase [1,2,3]. The influence of protein residues in shielding some of the reactive carbons must figure into the control of product regiochemistry and stereochemistry These two options relate directly to the studies we report here on the specificity of reaction by soybean lipoxygenase. Related observations in comparing the 9S-specific corn lipoxygenase with the 13S-specific soybean enzyme led to the important concept of substrate being capable of adopting a reversed orientation of binding in the lipoxygenase active site [18] This reversed orientation hypothesis has remained open to debate in that there is no LOX structure available with bound substrate to directly support or refute the various lines of argument. The aim of the present study was to investigate some novel aspects of the reaction of wild-type and Ala542 Gly mutant soybean LOX-1, including the specificity of the associated hydrogen abstraction and the characteristics of double dioxygenation as they pertain to our understanding of substrate binding and the stereocontrol of oxygenation

Objectives

Results

Conclusion