A fungal catalase reacts selectively with the 13S fatty acid hydroperoxide products of the adjacent lipoxygenase gene and exhibits 13S-hydroperoxide-dependent peroxidase activity

Abstract

The genome of the fungal plant pathogen Fusarium graminearum harbors six catalases, one of which has the sequence characteristics of a fatty acid peroxide-metabolizing catalase. We cloned and expressed this hemoprotein (designated as Fg-cat) along with its immediate neighbor, a 13S-lipoxygenase (cf. Brodhun et al, PloS One, e64919, 2013) that we considered might supply a fatty acid hydroperoxide substrate. Indeed, Fg-cat reacts abruptly with the 13S-hydroperoxide of linoleic acid (13S-HPODE) with an initial rate of 700–1300 s−1. By comparison there was no reaction with 9R- or 9S-HPODEs and extremely weak reaction with 13R-HPODE (~0.5% of the rate with 13S-HPODE). Although we considered Fg-cat as a candidate for the allene oxide synthase of the jasmonate pathway in fungi, the main product formed from 13S-HPODE was identified by UV, MS, and NMR as 9-oxo-10E-12,13-cis-epoxy-octadecenoic acid (with no traces of AOS activity). The corresponding analog is formed from the 13S-hydroperoxide of α-linolenic acid along with novel diepoxy-ketones and two C13 aldehyde derivatives, the reaction mechanisms of which are proposed. In a peroxidase assay monitoring the oxidation of ABTS, Fg-cat exhibited robust activity (kcat 550 s−1) using the 13S-hydroperoxy-C18 fatty acids as the oxidizing co-substrate. There was no detectable peroxidase activity using the corresponding 9S-hydroperoxides, nor with t-butyl hydroperoxide, and very weak activity with H2O2 or cumene hydroperoxide at micromolar concentrations of Fg-cat. Fg-cat and the associated lipoxygenase gene are present together in fungal genera Fusarium, Metarhizium and Fonsecaea and appear to constitute a partnership for oxidations in fungal metabolism or defense.

Oxidation of C18 Hydroxy‐Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase‐Related Hemoproteins Activated with Iodosylbenzene

“…As potential substrates for PhIO-activated Fg-cat, the 13 S -hydroxy derivatives of α-linolenic and γ-linolenic acids were selected for study because Fg-cat reacts specifically with the 13 S -hydroperoxides of unsaturated C18 fatty acids [14]. By itself Fg-cat is completely unreactive with the hydroxy derivatives, as is their treatment with PhIO alone (data not shown).…”

“…This P. fluorescens catalase (Pfl01-cat) has 32% sequence identity with Fg-cat and we have unpublished work establishing that it exhibits peroxidase activity using fatty acid 13 S -hydroperoxides as the oxidizing co-substrate (similar to Fg-cat [14]). When we tested the corresponding 13 S -hydroxy analogues as substrate for the Pfl01-cat in the presence of PhIO, the 18:3ω3 analogue was completely transformed to the 15,16-epoxide with only a trace of 13-ketone formation (Fig.…”

“…Three of the four catalase enzymes used in this study were cloned and expressed in E. coli as described previously: Fusarium graminearum gene FGSG_02217, encoding a 41.5 kD catalase-related hemoprotein designated here as Fg-cat [14], Mycobacterium avium ssp. paratuberculosis gene MAP_2744c, encoding a 33 kD mini-catalase [5] and human catalase was similarly expressed in E. coli [17].…”

“…paratuberculosis gene MAP_2744c, encoding a 33 kD mini-catalase [5] and human catalase was similarly expressed in E. coli [17]. The Pseudomonas fluorescens Pfl01 37.5 kD catalase (accession number WP_011333788.1) was cloned from the genomic DNA with an N-terminal His-tag (similarly to the other three catalases used in this study) and expressed in E. coli under the same conditions as used for Fg-cat [14]. The four catalases were purified by nickel affinity chromatography and quantified based on the absorbance of the main Soret band (at ~ 405 – 410 nm) assuming a molar extinction coefficient of 100,000 M −1 cm −1 .…”

“…When activated using PhIO, the P. homomalla cAOS catalyzed stereoselective epoxidations and hydroxylations of arachidonic acid and 8 R -hydroxy-eicosatetraenoic acid (the hydroxy analog of its natural 8R-hydroperoxy substrate) [9]. In the current work we further explored the oxidative capacity of catalase-related hemoproteins using three enzymes: Fg-cat, a 41 kD enzyme from Fusarium graminearum recently shown to act as a peroxidase using exclusively the 13 S -hydroperoxides of linoleic or α-linolenic acids [14]; a Pseudomonas fluorescens Pfl01 catalase (37.5 kD, accession number WP_011333788.1) with 32% sequence identity to Fg-cat; and the Mycobacterium avium ssp. paratuberculosis 33 kD catalase (gene MAB-2744c) previously shown to exhibit peroxidase activity using 13-hydroperoxy-linoleic acid (13-HPODE) as oxidizing co-substrate.…”