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. 2015 Aug;56(8):1606-15.
doi: 10.1194/jlr.M060178. Epub 2015 Jun 25.

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

Anneli Wennman  1 , Ann Magnuson  2 , Mats Hamberg  3 , Ernst H Oliw  1
Affiliations

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

Anneli Wennman et al. J Lipid Res. 2015 Aug.

Abstract

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-(2)H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the (2)H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.

Keywords: Fusarium gloeosporioides; Fusarium oxysporum; Pichia pastoris; gene expression; mass spectrometry; oxygenation mechanism; oxylipins; yeast expression.

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Figures

Fig. 1.
Fig. 1.
Overview of oxylipins formed by F. oxysporum and a phylogenetic tree of two LOX prototypes from soybean and coral and four fungal Mn- and FeLOXs. A: Overview of oxylipin biosynthesis by F. oxysporum. 9S-dioxygenase-allene oxide synthase (9S-DOX-AOS; FOXB_01332), 9R-dioxygenase (9R-DOX; FOXB_09952) (confirmed by recombinant expression; L. Sooman and E. H. Oliw, unpublished observations), and 10R-dioxygenase-epoxy alcohol synthase (10R-DOX-EAS; FOXB_03425) of distinct subfamilies with homology in their dioxygenase domains. The putative Fo-MnLOX has not been characterized, whereas FoxLOX is an iron 13S-LOX (11). B: Phylogenetic tree of informative sequences with homology to FoxLOX and Fo-MnLOX. Soybean 13S-LOX is often designated sLOX-1. The GenBank numbers are from top to bottom: FoxLOX (EXK38530), Pleurotis ostreatus (BAI99788), P. homomalla (ACC47283), Glycin max (P08170). F. oxysporum (EGU80482; FOXB_09004), C. gloeosporioides (EQB45907; CGLO_15145), and G. graminis (AAK81882). The GenBank accession number of an identical protein to FoxLOX is EXK38530; the accession locus of FoxLOX is FOXG_04807 at the Broad Institute.
Fig. 2.
Fig. 2.
SDS-PAGE of Fo-MnLOX samples from the purification procedure. Lanes from left to right: ladder, the growth medium, after capture of the enzyme by hydrophobic interaction chromatography (HIC), after gel filtration, and after deglycosylation with α-mannosidase and endoglycosidase H.
Fig. 3.
Fig. 3.
Normal phase HPLC-MS/MS analysis of oxidation of 18:2n-6 and 18:3n-3 to hydroperoxides by Fo- and Cg-MnLOX. A: Oxidation of 18:2n-6 and separation of products after reduction of hydroperoxides to alcohols with triphenylphosphine (TPP). Products formed by Fo-MnLOX (top) and by Cg-MnLOX (bottom). The labels 13, 11, and 9 indicate the corresponding HODEs. B: Oxidation of 18:3n-3 and separation of alcohols after reduction with TPP. Products formed by Fo-MnLOX (top) and by Cg-MnLOX (bottom). The labels 13, 11, and 9 indicate the corresponding hydroxyoctadecatrienoic acids. C:. Steric analysis by CP-HPLC-MS/MS analysis of 9-HODE (Chiralcel OB-H; 0.5 ml/min). D: Steric analysis by CP-HPLC-MS/MS analysis of 13-HODE formed by Fo-MnLOX (Reprosil Chiral AM; 0.2 ml/min). The elution order of the R and S stereoisomers of 13- and 9-HODE differ on Chiralcel OB-H and Reprosil Chiral AM. TIC, total ion current.
Fig. 4.
Fig. 4.
Analysis of products formed by Fo-MnLOX and oxidation of [11S-2H]18:2n-6 and Lα-lyso-glycero-3-phosphatidylcholine. A: Steric analysis of 11-HPODE. Incubation of 11-HPODE formed by Fo-MnLOX with 13R-MnLOX yielded 9R-HPODE as the main product and small amounts of racemic 13-HODE (analyzed on Reprosil Chiral AM after reduction with TPP: flow 0.2 ml/min). B: Separation of 11-HODE formed by Fo- and Cg-MnLOX (analyzed on Reprosil Chiral AM after reduction with TPP: flow 0.1 ml/min). Cg-MnLOX thus formed 11S-HPODE. The retention times of 11R-, 11S-, 13S-, and 9R-HODE under these conditions were 17.1, 19.1, 19.6 (not shown; right shoulder of 11S-HPODE), and 22.3 min, respectively. C: Oxidation of 18:2n-6 and [11S-2H]18:2n-6 by Fo-MnLOX. The activity is followed by measuring the increase in UV absorbance at 235 nm. The insert shows that the deuterium label is retained (zoom-scan MS analysis of HPODE after reduction to HODE with TPP). D: MS/MS spectrum of [11S-2H]11R-HODE formed from [11S-2H]18:2n-6 by Fo-MnLOX (after reduction with TPP). All but two fragments contained the deuterium label. Informative signals are present, among other things, at m/z 198 [−OOC-(CH2)8-CH=CH-CO2H; α-cleavage between C-11 and C-12), 180 (198-18; loss of water], and 154 (198-44; loss of CO2). E: Analysis of the rate of biosynthesis of cis-trans conjugated HPODE by Fo-MnLOX at different substrate concentrations. The deviation from Michaelis Menten kinetics might depend on biosynthesis of 11-HPODE (see text for details). F: Oxidation of Lα-lysoglycero-3-phosphatidylcholine (Lα-lysoGPC), dilinoleoyl-glycero-3-phosphatidylcholine (LL-GPC), and 18:2n-6 by Fo-MnLOX to products with UV absorbance at 235 nm. Lα-lysoglycero-3-phosphatidylcholine con­tained 47% 1-linoleoyl-2-hydroxy-glycero-3-phosphatidylcholine and a few percent 1-linoleneoyl-2-hydroxy-glycero-3-phosphatidylcholine, and the former was apparently oxidized.
Fig. 5.
Fig. 5.
X-band EPR spectra of Fo-MnLOX and metal analysis by ICP-AES. A: EPR spectra of recombinant Fo-MnLOX. The protein, as purified, revealed mainly a prominent Cu2+ signal (trace a). Denaturation by addition of concentrated H2SO4 revealed a strong and characteristic manganese sextet (Mn2+) on top of the Cu2+ signal (trace b). Spectrometer settings: temperature, 7 K; microwave power, 20 mW; modulation amplitude, 10 Gauss. B: ICP-AES analysis of recombinant and purified Fo-LOX and a blank sample from the final diafiltration step revealed 55 times more Mn than Fe. The metal analysis also confirmed the EPR analysis of Cu2+ and showed a high Cu content in relation to Mn of the protein sample.
Fig. 6.
Fig. 6.
CP-HPLC-MS analysis of oxidation products of 18:2n-6 formed by Fo-MnLOX·Ser348Phe. The products were reduced to alcohols with TPP and separated on Reprosil Chiral AM. A: Separation of stereoisomers of 13- and 9-HODE (flow 0.15 ml/min) in the same chromatogram. B: Separation of stereoisomers of 11-HODE (flow 0.1 ml/min). The top chromatogram represents analysis of 11-HODE formed by Fo-MnLOX·Ser348Phe. 11-HODE eluted in a single peak. The bottom chromatogram shows separation after addition of a small amount of 11R-HODE to the top sample and partial separation of stereoisomers.
Fig. 7.
Fig. 7.
A hypothetical model to illustrate the differences between Fo-MnLOX and Fo-MnLOX·Ser348Phe in the active site. A: Fo-MnLOX. B: Fo-MnLOX·Ser348Phe. The pentadiene unit (C-9 to C-13) of 18:2n-6 is positioned in the active sites for abstraction of the proR hydrogen in (A) and the proS hydrogen in (B).
Fig. 8.
Fig. 8.
Overview of the oxidation of 18:2n-6 to hydroperoxides by Fo-MnLOX and Fo-MnLOX·Ser348Phe. A: Catalytic profile of Fo-MnLOX. 11R- And 13S-HPODE are formed by suprafacial hydrogen abstraction and oxygen insertion. B: Hypothetical mechanism for Fo-MnLOX·Ser348Phe with abstraction of the proS hydrogen at C-11. The catalytic metal is marked by an orange sphere. The opposite head-to-tail substrate orientation in (B) compared with (A) seems unlikely, as both enzymes oxidized 1-linoleoyl-2-hydroxy-glycero-3-phosphatidylcholine.
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