Synthesis and Scavenging Role of Furan Fatty Acids
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Synthesis and scavenging role of furan fatty acids Rachelle A. S. Lemkea, Amelia C. Petersonb, Eva C. Ziegelhoffera, Michael S. Westphallc, Henrik Tjellströmd,e, Joshua J. Coonb,d,f, and Timothy J. Donohuea,d,1 Departments of aBacteriology, bChemistry, and fBiomolecular Chemistry, University of Wisconsin–Madison, Madison, WI 53706; dGreat Lakes Bioenergy Research Center, Madison, WI 53726; cGenome Center of Wisconsin, Madison, WI 53706; and eDepartment of Plant Biology, Michigan State University, East Lansing, MI 48824 Edited by Carol A. Gross, University of California, San Francisco, CA, and approved May 5, 2014 (received for review March 31, 2014) Fatty acids play important functional and protective roles in living Despite the proposed roles of Fu-FAs, little is known about systems. This paper reports on the synthesis of a previously how they are synthesized (13). We report on proteins needed for unidentified 19 carbon furan-containing fatty acid, 10,13-epoxy- the conversion of cis unsaturated fatty acids to 19Fu-FA. We 1 11-methyl-octadecadienoate (9-(3-methyl-5-pentylfuran-2-yl)nonanoic show that a O2-inducible protein (RSP2144) is an S-adenosyl me- acid) (19Fu-FA), in phospholipids from Rhodobacter sphaeroides. thionine (SAM)-dependent methylase that synthesizes a 19-carbon We show that 19Fu-FA accumulation is increased in cells contain- methylated trans unsaturated fatty acid (19M-UFA) from cis ing mutations that increase the transcriptional response of this vaccenic acid both in vivo and in vitro. We also identify gene 1 bacterium to singlet oxygen ( O2), a reactive oxygen species gen- products needed for the O2-dependent conversion of 19M-UFA 1 erated by energy transfer from one or more light-excited donors to 19Fu-FA. Further, we demonstrate that the presence of O2 to molecular oxygen. We identify a previously undescribed class leads to the disappearance of 19Fu-FA in vivo. Based on our of S-adenosylmethionine-dependent methylases that convert findings, we propose a pathway for Fu-FA synthesis and propose a phospholipid 18 carbon cis unsaturated fatty acyl chain to a 1 a protective role for compounds in the presence of a ROS like O2. 19 carbon methylated trans unsaturated fatty acyl chain (19M- UFA). We also identify genes required for the O2-dependent Results conversion of this 19M-UFA to 19Fu-FA. Finally, we show that Increased σE Activity Alters Cellular Fatty-Acid Composition. Fatty 1 . thepresenceof O2 leads to turnover of 19Fu-Fa in vivo We acids are targets for direct or indirect damage by ROS (1, 5–8, propose that furan-containing fatty acids like 19Fu-FA can act as 16), particularly when ROS are produced by integral membrane 1 a membrane-bound scavenger of O2, which is naturally produced enzymes in the respiratory chain or the photosynthetic apparatus by integral membrane enzymes of the R. sphaeroides photo- (1, 7, 8, 16, 18). The R. sphaeroides σE protein activates a tran- synthetic apparatus. 1 scriptional stress response to O2, a ROS that is generated by integral membrane proteins of the photosynthetic apparatus (16, radical scavenger | oxygenated fatty acid | fatty acyl methylase 17, 19). At least one ORF, which is a known member of the σE regulon, RSP2144, encodes a protein with amino acid simi- atty acids have crucial, yet diverse, roles in biology. In cells larity to an enzyme predicted to modify fatty acids (16, 17, 19– Fand organelles, fatty acids maintain bilayer stability, provide 21). To test for σE-dependent alterations in fatty acid composition, a permeability barrier, act as secondary messengers in signaling we prepared fatty acid methyl esters (FAMEs) to compare the pathways, and aid the function of integral membrane proteins fatty acid content of wild-type cells and mutant cells (ΔChrR; see (1–3). Fatty acids also help maintain viability in response to Table 1 for strain designations), which have high σE activity when temperature and environmental changes and can be targets for grown aerobically in the absence of light because the antisigma modification by reactive oxygen species or membrane-active – agents (2 8). Fatty acids, or the products derived from them, are Significance valuable as food additives, specialty chemicals, and petroleum substitutes (9–12). Thus, there is considerable interest in un- Fatty acids comprise a large class of compounds that serve derstanding the suite of fatty acids that can be made by native or broad roles in cells and society. These hydrophobic compounds engineered pathways. We are studying the synthesis and role of provide integrity for biological membranes, make them im- fatty acids during stress responses. permeable to solutes and toxins, and modulate the cellular Here, we demonstrate a previously unreported ability of the response to signals or stresses. Fatty acids, or the products photosynthetic bacterium Rhodobacter sphaeroides to produce derived from them, are also important as dietary supplements, furan-containing fatty acids (Fu-FAs), an important, yet poorly lubricants, specialty chemicals, and fuels. Their potential utility understood, class of compounds. The presence of Fu-FAs has in biology or industry could be increased by producing novel been reported previously in plants, fish, and some bacteria (13). classes of fatty acids. This paper reports on the occurrence and Based on their chemical properties, it is proposed that Fu-FAs synthesis of a newly discovered class of furan-containing fatty could provide bilayer protection against radicals or organic per- acid. It also provides evidence that furan-containing fatty acids oxides that reduce membrane function (13–15). The oxygen atom scavenge toxic reactive oxygen species, suggesting a pre- within Fu-FAs also provides a functional group for modifications viously unnoticed role for this class of compounds in bacteria that could increase their industrial value (13). and other cells. We discovered the 19-carbon furan-containing fatty acid 10,13- epoxy-11-methyl-octadecadienoate (9-(3-methyl-5-pentylfuran- Author contributions: R.A.S.L., A.C.P., E.C.Z., H.T., J.J.C., and T.J.D. designed research; 2-yl)nonanoic acid) (19Fu-FA) in phospholipids isolated from R.A.S.L., A.C.P., E.C.Z., M.S.W., and H.T. performed research; A.C.P., E.C.Z., M.S.W., H.T., and an R. sphaeroides mutant lacking an antisigma factor, ChrR, J.J.C. contributed new reagents/analytic tools; R.A.S.L., A.C.P., M.S.W., H.T., J.J.C., and T.J.D. that has increased transcription of genes that are normally acti- analyzed data; and R.A.S.L., A.C.P., E.C.Z., M.S.W., H.T., J.J.C., and T.J.D. wrote the paper. vated in the presence of the reactive oxygen species (ROS) The authors declare no conflict of interest. 1 1 singlet oxygen ( O2). In this and other phototrophs, O2 is This article is a PNAS Direct Submission. a byproduct of light energy capture in integral membrane com- Freely available online through the PNAS open access option. plexes of the photosynthetic apparatus (5, 16, 17). Consequently, 1To whom correspondence should be addressed. Email: [email protected]. fatty acids or other membrane components are likely targets for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1 damage by O2 (16, 17). 1073/pnas.1405520111/-/DCSupplemental. E3450–E3457 | PNAS | Published online August 4, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1405520111 Downloaded by guest on September 23, 2021 Table 1. Strains and plasmids PNAS PLUS Strains/plasmids Relevant genotype Source Strains E. coli DH5α supE44 lacu169(Ф80 lacZ M15) hsdR178 recA1 endA1 gyrA96 thi-1 relA-1 (52) S17-1 C600::RP-4 2-(Tc::Mu) (Kn::Tn7) thi pro hsdR Hsd M+recA (53) BL21(DE3) F− ompT hsdSB (rB- mB-) gal dcm (DE3) Novagen JW1653 cfa::kan of BW25113 Keio Collection (48) RLcfaK49-6 cfa markerless deletion mutant of JW1653 This study R. sphaeroides 2.4.1 Wild type (36) TF18 rpoE::drf (23) ΔChrR chrR::drf (54) ΔRSP2144 RSP2144::Ω SmrSpr (20) RSL1 ΔchrR RSP2144::Ω SmrSpr This study 1091:spR/ΔChrR ΩSpR insertion in RSP1091 coding sequence in ΔChrR This study Delta ΔRSP1091/ ΔChrR In-frame deletion of both RSP1091 and ChrR This study Plasmids pBlueScriptII KS- Apr Agilent Technologies pRS2144 RSP2144 in pBSII (20) r pET-28a+ His6 expression vector, Kn Novagen pRLhisRSP2144 1.2-kb RSP2144 fragment from pRS44 cloned into NdeI/EcoRI-cut pET-28a This study pIND5 pIND4 NcoI site replaced with NdeI site, Knr (20) pRL101 1.3-kb fragment amplified from pRLhisRSP2144 cloned into NdeI/HindIII pIND5 This study pAYW19 E. coli cfa gene on pGEM5, Apr (49) MICROBIOLOGY factor ChrR that normally inhibits σE function has been inacti- mulates in cells with increased σE activity, shows that it has an vated (19, 20, 22, 23). intact molecular ion mass of 322.2502 Da, corresponding to a In wild-type cells, we found the expected major FAME prod- molecular formula of C20H34O3 (Fig. 2A). The fragmentation pat- ucts (C18:1, C18:0, C16:1, C16:0) (Table 2), based on published tern has good correlation with a methyl ester of a 19-carbon furan- fatty-acid analysis of R. sphaeroides (24–27). In ΔChrR cells, we containing fatty acid, 10,13-epoxy-11-methyl-octadecadienoate observed the accumulation of two additional FAME products (9-(3-methyl-5-pentylfuran-2-yl)nonanoic acid), as seen by the (retention times of ∼16.4 and 17.5 min in Fig. 1) and lower levels comparison with the reference spectrum in Fig. 2A [spectrum of the vaccenic acid (C18:1) FAME compared with wild-type M11703; American Oil Chemists’ Society (AOCS) Lipid Library].