Enzymatic Synthesis of a Bicyclobutane Fatty Acid by a Hemoprotein–Lipoxygenase Fusion Protein from the Cyanobacterium Anabaena PCC 7120

Enzymatic synthesis of a bicyclobutane fatty acid by a hemoprotein–lipoxygenase fusion protein from the cyanobacterium Anabaena PCC 7120

Claus Schneider*, Katrin Niisuke*, William E. Boeglin*, Markus Voehler†, Donald F. Stec†, Ned A. Porter†, and Alan R. Brash*‡

Departments of *Pharmacology and †Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232

Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved October 5, 2007 (received for review July 30, 2007) Biological transformations of polyunsaturated fatty acids often that the catalase-related allene oxide synthase (AOS) is proto- lead to chemically unstable products, such as the prostaglandin typical of an enzyme family that also has diversified functions. endoperoxides and leukotriene A4 epoxide of mammalian biology Accordingly, the unusual catalytic activity of the catalase-related and the allene epoxides of plants. Here, we report on the enzy- coral AOS provided the impetus for the present investigation, matic production of a fatty acid containing a highly strained bicyclic namely to explore other possible occurrences of catalase-related four-carbon ring, a moiety known previously only as a model proteins with novel functions in the biotransformation of poly- compound for mechanistic studies in chemistry. Starting from unsaturated fatty acids. linolenic acid (C18.3␻3), a dual function protein from the cyanobac- By using BLAST searches for sequences similar to the coral terium Anabaena PCC 7120 forms 9R-hydroperoxy-C18.3␻3ina catalase-related domain, one of the top matching hits besides lipoxygenase domain, then a catalase-related domain converts the other coral homologues was identified in the cyanobacterium 9R-hydroperoxide to two unstable allylic epoxides. We isolated Anabaena sp. strain PCC 7120. The Anabaena genus of cya- and identified the major product as 9R,10R-epoxy-11trans-C18.1 nobacteria are photosynthetic prokaryotes that grow in long containing a bicyclo[1.1.0]butyl ring on carbons 13–16, and the strings or filaments and that can develop a nitrogen-fixing ability minor product as 9R,10R-epoxy-11trans,13trans,15cis-C18.␻3, an in specialized heterocysts. They are studied as a model for epoxide of the leukotriene A type. Synthesis of both epoxides can prokaryotic developmental biology (12). Anabaena PCC 7120 be understood by initial transformation of the hydroperoxide to an has a genome of 6.4 Mb, and the cells also contain several large epoxy allylic carbocation. Rearrangement to an intermediate bicy- plasmids. The novel gene resides on the 102 kb gamma plasmid. clobutonium ion followed by deprotonation gives the bicyclobu- Enticingly, this small catalase-related sequence was found in the tane fatty acid. This enzymatic reaction has no parallel in aqueous same ORF as a LOX-like sequence, albeit a highly unusual one, or organic solvent, where ring-opened cyclopropanes, cyclobu- much smaller than any previously known member of the LOX tanes, and homoallyl products are formed. Given the capability superfamily. In a separate study, we show that this C-terminal shown here for enzymatic formation of the highly strained and domain of the fusion protein is a catalytically complete lipoxy- unstable bicyclobutane, our findings suggest that other transfor- genase that specifically forms 9R-hydroperoxides from C18 mations involving carbocation rearrangement, in both chemistry polyunsaturated fatty acid substrates (Y. Zheng, W.E.B., C.S., and biology, should be examined for the production of the high A.R.B., unpublished data). Here, we report characterization of energy bicyclobutanes. the catalytic activities of the N terminus of the fusion protein, the heme-containing domain with sequence similarity to catalase. catalase ͉ carbocation ͉ epoxide ͉ leukotriene ͉ bicyclobutonium ion This unusual enzyme utilizes the 9R-hydroperoxylinolenic acid (C18.3␻3) product of the LOX domain as a substrate and he ability of lipoxygenase (LOX) enzymes to oxygenate converts it to two epoxy fatty acids, the major one of which Tpolyunsaturated fatty acids to specific fatty acid hydroper- contains a bicyclic four-carbon ring. Its synthesis has important oxides is used throughout the eukaryotic world for the produc- implications for the possible existence of novel carbocation tion of signaling molecules and other complex products (1–4). rearrangements in both chemistry and biology. The initial hydroperoxy fatty acid product is often further transformed to a highly unstable biosynthetic intermediate. Results Thus, plants express specialized cytochrome P450 enzymes of Protein Sequences and Alignments. The novel hemoprotein from the CYP74 family that convert hydroperoxy-C18 fatty acids to Anabaena and the AOS domain from the coral Plexaura homomalla allene oxides, the best characterized of which is an intermediate share an overall 35% amino acid identity. Particularly significant in biosynthesis of the hormone jasmonic acid (5). In the leuko- matches are conserved around the distal heme His residue and the cytes of higher animals, the 5-LOX enzyme forms the initial distal heme Asn [supporting information (SI) Fig. 5]. A very 5-hydroperoxy-C20.4 product and converts it into the highly significant mismatch occurs around the heme proximal ligand, unstable epoxide leukotriene A4 (LTA4), from which the other leukotriene family members arise (6). As yet another facet of this theme, marine corals express a natural fusion protein (7) in Author contributions: A.R.B. designed research; C.S., K.N., W.E.B., M.V., and D.F.S. per- CHEMISTRY formed research; C.S., K.N., W.E.B., M.V., D.F.S., N.A.P., and A.R.B. analyzed data; and C.S., which a LOX domain converts arachidonic acid to its 8R- N.A.P., and A.R.B. wrote the paper. hydroperoxide and a catalase-related domain effects a further The authors declare no conflict of interest. transformation to an unstable allene oxide, a potential interme- This article is a PNAS Direct Submission. diate in formation of marine prostanoids (8). This catalase- ‡To whom correspondence should be addressed at: Department of Pharmacology, Vander- related domain of the coral fusion protein is structurally similar bilt University School of Medicine, 23rd Avenue South at Pierce, Nashville, TN 37232-6602. to true catalases (9) yet quite distinct in function. Based on the E-mail: [email protected]. knowledge that the plant CYP74 enzyme family exhibits a This article contains supporting information online at www.pnas.org/cgi/content/full/

spectrum of catalytic reactions, including formation of allene 0707148104/DC1. BIOCHEMISTRY oxides, aldehydes, or vinyl ethers (10, 11), there is the possibility © 2007 by The National Academy of Sciences of the USA

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Fig. 1. Preparation, puriﬁcation, and UV analysis of unstable epoxides. An ice-cold solution of 9R-hydroperoxylinolenic acid (90 ␮M) in 10 ml of ice-cold hexane was vortex-mixed for 2 min with Anabaena catalase–LOX enzyme (0.8 nmol) in 0.2 ml of phosphate buffer, pH 8; reaction with the same sample of enzyme was repeated twice more using fresh substrate in ice-cold hexane. The combined hexane phases were evaporated to Ϸ2 ml under a strong stream of nitrogen, treated with diazomethane and 1% ethanol for 10 s at 0°C, and then evaporated to dryness and stored in hexane at Ϫ70°C. (A) UV spectrum of the hydroperoxy substrate in hexane before reaction and the hexane phase after mixing with enzyme. (B) Reversed-phase HPLC analysis of the product methyl esters with UV detection at 205 and 270 nm. A Waters Symmetry C18 column (25 ϫ 0.46 cm) was eluted with methanol/20 mM aqueous triethylamine at pH 8 [80:20 (vol/vol)] at a ﬂow rate of 1 ml/min. (C) Normalized UV spectra of the two main products.

which is a Tyr in the coral AOS, as is characteristic of all catalase mophores, one near 200 nm and the other with ␭max at 278 nm family members, yet by alignment this residue is replaced by His in characteristic of the leukotriene A class of allylic epoxides (Fig. Anabaena. Remarkably, therefore, the Anabaena sequence appears 1A) (18). The resulting hexane extract was treated with dia- to represent a His-ligated heme in the context of a catalase-related zomethane for 10 s at 0°C to form the methyl ester derivative (19) protein framework. We should note that, whereas Anabaena is a and the fatty acid derivatives subsequently analyzed by reversed- prokaryotic cyanobacterium and other cyanobacteria are found as phase HPLC using conditions adapted from a method for symbionts in corals (13), the P. homomalla AOS–LOX is unam- analysis of synthetic leukotriene A4 (Fig. 1B) (20). HPLC biguously eukaryotic based on the presence of multiple introns in analysis showed near quantitative conversion to two products, the DNA (unpublished observations). Nonetheless, such coexist- present in a 2:1 ratio as determined by using a 14C substrate. The ence could have provided the opportunity for an earlier gene more prominent product 1 displays a UV spectrum with end transfer one way or the other. absorbance extending beyond 230 nm, and product 2 has the spectrum of a conjugated triene, ␭max 278 nm (Fig. 1C). LC-MS Expression and Purification of the Anabaena Fusion Protein. We analysis using positive ion electrospray ionization revealed that expressed the whole Anabaena fusion protein as well as the the two products have the same molecular weight (306 for the isolated LOX domain in Escherichia coli and partially purified methyl ester) as indicated by their identical adduct ions with the proteins by nickel affinity chromatography by using N- sodium, potassium, and triethylamine. terminal His6-tags; (expression of the catalase-related domain by itself gave protein containing no heme and exhibiting no cata- Identification of the Major Allylic Epoxide Product. The 1H-NMR lytic activity). Anabaena contains abundant polyunsaturated spectrum of product 1 methyl ester, together with the COSY fatty acids and is particularly rich in linolenic acid (C18.3␻3) analysis of cross-couplings, outlined a structure of a 9,10trans- (14–16). We found that this fatty acid is oxygenated by the LOX epoxy-11trans-C18.1 derivative (Fig. 2 and SI Dataset 1). With domain to the corresponding 9R-hydroperoxide (a contrast with only one double bond and yet the same molecular weight as the plant 9-LOX enzymes, which have S stereospecificity) (5). The leukotriene A-type epoxide, it follows that the structure of 1 lipoxygenase is not involved in further metabolism, a point we must contain two rings. The arrangement of the carbon atoms established by using the separately expressed LOX domain. was further investigated through conventional proton–proton However, the catalase-related domain of the fusion protein decoupling experiments, heteronuclear single quantum correla- avidly metabolizes the 9R-hydroperoxide to a complex spectrum tion (HSQC), and heteronuclear multiple bond correlation of stable end products, including triols, diols, and epoxyalcohols (HMBC) to identify the carbon–proton couplings, the distor- (data not shown). It appeared likely that the primary enzymic tionless enhancement polarization transfer (DEPT) experiment product of the catalase-like hemoprotein is an unstable epoxide to confirm the CH2, CH, and C carbons, and NOESY for or epoxides, and we reasoned that if this could be analyzed through-space couplings (SI Figs. 6–9). The key structural data directly it would greatly simplify the product profile and help for establishing the bicyclobutane ring was the fact that all four clarify the fundamental mechanism of biosynthesis. carbons of C13 through C16 appeared as CH signals in the HSQC and DEPT experiments, suggesting that, given their Isolation of Two Allylic Epoxides Formed by the Catalase-Related chemical shift values, each of these carbons is bound to three Domain. We explored conditions under which the Anabaena other carbons and one proton. The C14 and C15 carbons, which enzyme reacted with pure 9R-hydroperoxylinolenic acid in a exhibit almost identical chemical shifts at 9.3 ppm, are clearly biphasic hexane/pH 8 aqueous system that would simultaneously resolved in the carbon spectrum at 800 MHz (SI Fig. 8). The extract the hydroperoxy substrate from the hexane into the COSY analysis showed strong correlation among the proton water, allow enzymic metabolism, and then instantly extract the signals of H14, H15, and H16 of the ring and extending to H17 less polar epoxide product(s) back into the hexane, thus afford- and H18 of the ␻-carbon chain. H13 (a doublet at 2.36 ppm) ing protection from hydrolysis, an approach similar to the one we couples only to H12 on the double bond; its couplings with the developed for isolation of allene oxides (17). After 2 min of neighboring protons H14 and H15 thus must be very small, vortex mixing at 0°C, UV spectroscopy of the hexane showed indicating that H14 and H15 are held at almost right angles in the disappearance of substrate and appearance of new chro- bicyclic ring system. The through space cross-peaks in the

18942 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0707148104 Schneider et al. Downloaded by guest on September 25, 2021 H14 210128 H16 18 H3CO2C 9 O 11 H13 H15 2 13 10 9 -OCH3

17 18 14,15 4, 5, 3 8 6 16 7a,b

2 14 17 18 -OCH 3 15 4, Fig. 3. Conﬁguration of the bicyclobutane ring of product 1. The NOESY 12 11 5, 10 13 8 NMR spectrum of product 1 was recorded in d6-benzene at 600 MHz. The 9 3 6 16 7 partial chemical structure illustrates the through-space couplings of the single protons at H13 and H16, the two ends of the bicyclobutyl ring. The 13-exo,16- integration: 2 3 1 1 121 2 626 3 endo conﬁguration of the ring can be deduced from the observation that the coupling of H13 to H14/15 is weak, whereas H16-H14/15 is strong, and that the coupling of H13 to H17 is strong, whereas there is no detectable NOE between 3,4 H12 and H16 (SI Fig. 9). 8,9 17,18

2,3 allylic epoxide with a conjugated triene double bond system (SI 16,17 16,14/15 Dataset 2). Based on the coupling constants, the configuration 12,13 9,10 of the 11,12, 13,14, and 15,16 double bonds is defined as trans,trans,cis (J ϭ 15.2, 14.8, and 11.0 Hz, respectively). Con- 10,11 sidering also that the configuration of C9 should not change during the transformation of the 9Rhydroperoxide to the 9,10- epoxide, product 2 was identified as a leukotriene A-type epoxide, 9R,10R-trans-epoxyoctadeca-11E,13E,15Z-trienoic acid. This structure is directly analogous to the structure of the mammalian 5-LOX product, LTA4,a5S,6S-trans-epoxy-20.4␻6 fatty acid with a trans,trans,cis (7E,9E,11Z) conjugated triene. Interestingly, biosynthetically produced LTA4 has not been subject to a complete and direct structural analysis. Knowledge of its structure rests on comparison of its biotransformation and hydrolytic reactions with synthetic LTA4, and on an understand- Fig. 2. NMR analysis of product 1. Spectra were recorded in d6-benzene at ing of its mechanism of biosynthesis by the leukocyte 5-LOX 283 K using a Bruker 600 MHz spectrometer equipped with a cryoprobe. The enzyme (21, 22). two-dimensional H,H COSY spectrum is shown below, with an expanded view of two areas of the spectrum depicted above. On the COSY spectrum, the Discussion correlations are clear from H8 through the trans epoxide (H9, H10, J ϭ 2.0 Hz) to the trans double bond (H11, H12, J ϭ 15.5 Hz) to H13 of the bicyclobutane Bicyclobutane is the simplest bicyclic hydrocarbon (Scheme 1). ring, a doublet at 2.36 ppm. H14 and H15 are in a very similar chemical It has a bond strain energy of 66 kcal/mol, more than double the environment and, therefore, have similar chemical shifts in the proton (1.29– value of either cyclopropane or cyclobutane (Ϸ26 kcal/mol) (23). 1.36 ppm) and carbon (9.2 and 9.3 ppm) spectra (SI Fig. 8). Both H14 and H15 Extensive efforts have been made during the past 40 years to are coupled to H16, which extends the correlation into the ethyl substituent develop synthetic approaches to molecules containing a bicy- (H17 and H18). Although the correlations through H13 are not evident here, clobutane subunit and to understand the chemistry of this H12 shows a three-bond correlation to both C14 and C15 in the HMBC structure (24–27). Although bicyclobutanes have been the focus spectrum (SI Fig. 7). CHEMISTRY

NOESY spectrum confirm the covalent structure and confirmed H assignment of the configuration of the side chains (C1–C12, C17, H and C18) as exo–endo (Fig. 3). Thus, the complete covalent structure of product 1 was established as 9R,10Rtrans- H H epoxyoctadeca-11trans-(13,14,15,16-bicyclo[1.1.0]butyl)-enoic acid. H H

1 BIOCHEMISTRY Identification of Product 2 as a LTA4 Analogue. The H-NMR and H,H-COSY spectra of product 2 established the structure of an Scheme 1. Bicyclo [1.1.D] butane.

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Fig. 4. Transformation of 9R-hydroperoxy-C18.3␻3bytheAnabaena catalase-related enzyme.

of many fundamental studies in organic chemistry, this structure and we isolated a single bicyclobutyl isomer, arguing for stringent is, to our knowledge, heretofore unknown in nature. We report reaction control in the enzyme active site. here on the isolation and identification of a substituted bicy- Historically, the catalase gene family is known for its role in clobutane formed in biosynthesis from a fatty acid hydroperox- protection from oxidative stress via the breakdown of hydrogen ide precursor. The proposed biosynthetic mechanism proceeds peroxide, yet clearly the Anabaena gene has a biosynthetic through the rearrangements of carbocations and is completed function. A role in biosynthesis is precedented by the catalase- with an enzymatic step of deprotonation of a bicyclobutonium related domain of the coral AOS–LOX fusion protein that ion yielding a bicyclobutane ring via a route not observed in converts 8R-hydroperoxy-arachidonic acid to an allene oxide, a solution chemistry. reaction equivalent to that of the P450 AOS of plants (10). With Transformation of the linolenate hydroperoxide to the allylic the recent successful x-ray structural analysis of the coral cata- epoxides 1 and 2 can be understood by a mechanism involving lase-related domain, its modest sequence similarity to true intermediate carbocations (Fig. 4). The conversion of conju- catalases evolved into remarkable parallels in three-dimensional gated diene hydroperoxides to allyl carbocations like 3 has ample structure (9). The heme-binding pocket and surrounding protein precedent, being implicated in the enzymatic synthesis of allene network of true catalases are well conserved, whereas the oxides, vinyl ethers, and other fatty acid derivatives (28), as well structural features involved in melding together the catalase as related transformations of prostaglandin endoperoxides to homotetramer are absent. The coral AOS domain is 43 kDa in size and crystallizes as a dimer (9), whereas true catalases usually prostacyclin and thromboxane A2 (29). Removal of a proton from the carbocation 3 gives the leukotriene-type epoxide, are tetrameric or hexameric with subunits of 55–69 kDa product 2. Although the structure of 2 is directly analogous to (‘‘small’’) or 75–84 kDa (‘‘large’’) (39). The Anabaena hemoprotein domain is Ϸ41 kDa in size, with strong homology to the that of the leukocyte 5-LOX product LTA4, its mechanism of biosynthesis is quite different. Whereas epoxide 2 is formed via coral AOS, except near the end of the sequence where the proximal heme ligand appears to be substituted with a histidine initial activation of the hydroperoxide and subsequent rear- (SI Fig. 5). The 9R-hydroxy metabolites of linolenic and linoleic rangements of an epoxy allylic carbocation with a final elimina- ϩ acids have been detected in Ϸ5:1 ratio in extracts of a species of tion of H (Fig. 4), the biosynthesis of LTA involves an initial 4 Anabaena (40), but these organisms have yet to be examined for LOX-catalyzed hydrogen abstraction from the carbon chain, the more complex products that might be expected to arise from followed by radical rearrangements that lead to cleavage of the the epoxy-bicyclobutyl linolenate. The Anabaena hemoprotein– hydroperoxide and epoxide formation at the ultimate step. lipoxygenase fusion protein resides on the ␥-plasmid in Carbocation 3 is homoallylic and, as such, provides access to Anabaena PCC 7120, and, by analogy with the plasmid-encoded the cyclopropylcarbinyl and bicyclobutonium ions (30), two of antibiotic resistance genes, it may have a specialized role and which are shown in Fig. 4. The nature of these carbocation confer an advantage in a selected environment. Other small intermediates has been an important chapter in the ‘‘classical– catalase analogues reside in the genomes of many microorgan- nonclassical’’ ion debate (31, 32). It is generally agreed that the isms and have the potential for additional functions in the most stable carbocation intermediates present are separated by metabolism of natural peroxides. low-energy barriers (33) and that cyclopropanes, cyclobutanes, Given the capability shown here for enzymatic transformation to and homoallylic structures are the predictable set of products the highly strained and unstable bicyclobutane, other enzymic that form from these intermediates and the equivalent alcohol transformations involving carbocation rearrangements should be derivatives in aqueous media (34). Such reactions occur in examined for the production of the high-energy bicyclobutanes. enzymatic transformations to a wide array of natural products Immediate extraction of unstable reaction products may also be (e.g., refs. 28 and 35–38). In the case of the Anabaena catalase- applicable to purely chemical transformations and may allow for the related protein, we suggest that generation of bicyclobutonium isolation of products believed not to be existent or considered too ion 5 in the vicinity of an enzymatic base provides an additional unstable for characterization. We show that, with appropriate product-forming route, deprotonation to give the bicyclobutane, methodology, even previously intractable biological products, such product 1. This is a most unusual outcome that is not reproduced as the leukotriene A-type epoxide, and novel ones, like the bicy- in solution chemistry. The enzyme must provide an environment clobutane, are amenable to recovery and structural analysis. in which proton abstraction from the bicyclobutonium ion facilitates production of the high-energy bicyclobutyl moiety, a Materials and Methods species we then recovered by its immediate extraction into Materials. Fatty acids was purchased from NuChek Prep. hexane. Notably, a potential epoxide product arising via proton [1-14C]Linolenic acid was purchased from NEN Life Science abstraction from the cyclopropylcarbinyl ion 4 was not detected, Products.

18944 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0707148104 Schneider et al. Downloaded by guest on September 25, 2021 Cloning, Expression, and Purification of Anabaena Enzyme Constructs. of reaction at a higher concentration of substrate (100–250 ␮M), The cDNA for the full-length fusion protein was cloned by PCR reactions were conducted in a 2-mm path-length microcuvette. from Anabaena sp. strain PCC 7120 genomic DNA, a kind gift from James W. Golden (Texas A&M University, College Sta- Extraction and HPLC Analysis of Unstable Products. Enzyme reactions tion, TX). Sequencing confirmed the identity to the published were conducted at 0°C, with the substrate initially in hexane (10 ml, sequence in the National Center for Biotechnology Information Ϸ100 ␮M 9-hydroperoxide) layered over the Anabaena enzyme database (NP࿝478445) and at CyanoBase (http://bacteria. (0.8 nmol) in 200 ␮l of phosphate buffer, pH 8. The reaction was kazusa.or.jp/cyanobase). Several different cDNA expression initiated by vigorous vortex mixing of the two phases, which was constructs were prepared: each listed below was cloned into continued for 2 min; then the test tube was placed back on ice. The pET17b for expression in E. coli. Full-length constructs included hexane phase was scanned from 200–350 nm in UV light by using the native cDNA sequence (amino acid code MDLNTY— a Perkin-Elmer Lambda-35 spectrophotometer, and, if all substrate LMMSINI.) and the same sequence with a His tagontheN was consumed (as in Fig. 1A), the reaction was repeated with fresh 6 substrate in hexane mixed with the same batch of enzyme. The terminus (MHHHHHHDLNTY—LMMSINI.). These con- combined hexane phases were evaporated to Ϸ2mlbyusinga structs expressed with similar heme content (with the main Soret vigorous stream of nitrogen. The sample was then treated with band observed at 406 nm) and were used for enzyme quantifi- ␮ ␧ ϭ ethanol (20 l) and ethereal diazomethane for 10 s at 0°C and then cation assuming 100,000. The affinity-purified His-tagged rapidly evaporated to dryness and stored in hexane at Ϫ20°C until expression construct was used throughout these experiments. further analysis. The same procedures but omitting the methylation The proteins were expressed in E. coli BL21 (DE3) cells (No- step gave samples of the free acids. vagen) using methodology we described previously (41), and the The presence of an excess of alcohol over water in a slightly His6-tagged protein was purified on Ni-NTA agarose (Qiagen) basic solution greatly prolongs the half-life of allylic epoxides, according to the manufacturer’s instructions. The lipoxygenase- such as leukotriene A4, allowing their analysis by reversed phase only domain (starting at amino acid 344 with the amino acid HPLC at room temperature (20). The hexane extracts were sequence KDDLPGK. . . and comprising the last 430 aa of the analyzed and purified by using a Waters Symmetry C18 5-␮m full-length construct) was expressed with an N-terminal His6 tag column (0.46 ϫ 25 cm) eluted at a flow rate of 1 ml/min with and purified by nickel affinity chromatography. We also at- methanol/20 mM potassium phosphate, pH 8 (replaced with tempted to express several constructs encoding the N-terminal triethylamine for LC-MS analysis; see below), in the proportions domain only (amino acids 1–344). These constructs had the His 80:20 (vol/vol), with UV light detection at 205, 220, 235, and 270 tag placed at either the N or C terminus (or no His tag) and nm using an Agilent 1100 series diode array detector. The main included constructs with the C terminus extended a further 20 aa products were recovered by extraction with cold hexane followed along the fusion protein. However, these constructs expressed by evaporation to dryness under a strong stream of nitrogen. with no heme and exhibited no catalytic activity. LC-MS and NMR analysis. LC-MS of the allylic epoxides was Preparation of Hydroperoxides. 9R-Hydroperoxy-C18.3␻3 was pre- performed using a Thermo Finnigan LC Quantum instrument. ϫ pared by using the LOX domain of the Anabaena enzymeinpH A Waters Symmetry C18 column (0.2 15 cm) was eluted with 7.5 Tris buffer according to the methods described previously for methanol/20 mM aqueous triethylamine adjusted to pH 8 with other fatty acid hydroperoxides (42). The product was extracted acetic acid [80:20 (vol/vol)] at 0.2 ml/min. The heated capillary into dichloromethane and purified by SP-HPLC (Beckman 5␮ ion lens was operated at 220°C. Nitrogen was used as a nebuli- silica column; 25 ϫ 0.46 cm) using a solvent system of hexane/ zation and desolvation gas. The electrospray potential was held at 4 kV. Source-induced dissociation was set at Ϫ10 eV. Mass isopropanol/acetic acid 100:1:0.1 by volume (flow rate of 1 spectra were acquired over the mass range m/z 100–500 at2sper ml/min), with detection of the product by UV detection at 235 scan. Collision-induced dissociation was performed at Ϫ15 eV. nm (42). The product was quantified by UV spectroscopy (␧ ϭ 13 Ϫ NMR spectra were recorded on a Bruker 800-MHz ( C and 23,000 at 235 nm) and stored at 20°C in ethanol. DEPT) or 600-MHz instrument at 283 K. The samples were dissolved in d6-benzene, and the chemical shifts are reported Activity Assays. Small-scale incubations with the purified enzymes relative to the benzene signal (␦ 7.16 ppm for hydrogen and 128.0 were typically conducted in a 0.5-ml UV cuvette and analyzed by ppm for carbon). Both instruments were equipped with a Bruker UV spectrometry in an incubation buffer (50 mM Tris, 150 mM TCI cryoprobe. NaCl, pH 7.5). Enzyme activity was monitored by repetitive scan- ning in the range 350–200 nm or by monitoring disappearance of We thank Dr. Thomas M. Harris for careful review of the NMR data. This the signal at 235 nm in the time-drive mode. To measure the rate work was supported by National Institutes of Health Grant GM-74888.

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