Purification and Catalytic Activities of the Two Domains of the Allene Oxide Synthase-Lipoxygenase Fusion Protein of the CoralPlexaura homomalla

Abstract

The conversion of fatty acid hydroperoxides to allene epoxides is catalyzed by a cytochrome P450 in plants and, in coral, by a 43-kDa catalase-related hemoprotein fused to the lipoxygenase that synthesizes the 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) substrate. We have expressed the separate lipoxygenase and allene oxide synthase (AOS) domains of the coral protein in Escherichia coli (BL21 cells) and purified the proteins; this system gives high expression (1.5 and 0.3 μmol/liter, respectively) of catalytically active enzymes. Both domains show fast reaction kinetics. Catalytic activity of the lipoxygenase domain is stimulated 5-fold by high concentrations of monovalent cations (500 mmNa+, Li+, or K+), and an additional 5-fold by 10 mm Ca2+. The resulting rates of reaction are ≈300 turnovers/s, 1–2 orders of magnitude faster than mammalian lipoxygenases. This makes the coral lipoxygenase well suited for partnership with the AOS domain, which shows maximum rates of ≈1400 turnovers/s in the conversion of 8R-HPETE to the allene oxide. Some unusual catalytic activities of the two domains are described. The lipoxygenase domain converts 20.3ω6 partly to the bis-allylic hydroperoxide (10-hydroperoxyeicosa-8,11,14-trienoic acid). Metabolism of the preferred substrate of the AOS domain, 8R-HPETE, is inhibited by the enantiomer 8S-HPETE. Although the AOS domain has homology to catalase in primary structure, it is completely lacking in catalatic action on H2O2; catalase itself, as expected from its preference for small hydroperoxides, is ineffective in allene oxide synthesis from 8R-HPETE. The conversion of fatty acid hydroperoxides to allene epoxides is catalyzed by a cytochrome P450 in plants and, in coral, by a 43-kDa catalase-related hemoprotein fused to the lipoxygenase that synthesizes the 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) substrate. We have expressed the separate lipoxygenase and allene oxide synthase (AOS) domains of the coral protein in Escherichia coli (BL21 cells) and purified the proteins; this system gives high expression (1.5 and 0.3 μmol/liter, respectively) of catalytically active enzymes. Both domains show fast reaction kinetics. Catalytic activity of the lipoxygenase domain is stimulated 5-fold by high concentrations of monovalent cations (500 mmNa+, Li+, or K+), and an additional 5-fold by 10 mm Ca2+. The resulting rates of reaction are ≈300 turnovers/s, 1–2 orders of magnitude faster than mammalian lipoxygenases. This makes the coral lipoxygenase well suited for partnership with the AOS domain, which shows maximum rates of ≈1400 turnovers/s in the conversion of 8R-HPETE to the allene oxide. Some unusual catalytic activities of the two domains are described. The lipoxygenase domain converts 20.3ω6 partly to the bis-allylic hydroperoxide (10-hydroperoxyeicosa-8,11,14-trienoic acid). Metabolism of the preferred substrate of the AOS domain, 8R-HPETE, is inhibited by the enantiomer 8S-HPETE. Although the AOS domain has homology to catalase in primary structure, it is completely lacking in catalatic action on H2O2; catalase itself, as expected from its preference for small hydroperoxides, is ineffective in allene oxide synthesis from 8R-HPETE. allene oxide synthase hydro(pero)xyeicosatetraenoic acid reversed-phase high pressure liquid chromatography high pressure liquid chromatography polymerase chain reaction gas chromatography-mass spectrometry trimethylsilyl lipoxygenase polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid absorbance unit nitrilotriacetic acid The Caribbean coral Plexaura homomalla is famous for its high content of prostaglandin esters, which constitute 2-3% of the coral dry weight (1Bundy G.L. Adv. Prostaglandin Thromboxane Leukotriene Res. 1985; 14: 229-262PubMed Google Scholar). In the early 1970s, at a time when practical chemical syntheses of the prostaglandins were still under development,P. homomalla was used as a commercial source of these mammalian hormones. This practical interest led in turn to investigation of the coral prostaglandin synthase. The issue of the biochemical pathway turned out to be difficult to tackle in P. homomalla itself, because, for reasons that remain unclear, prostaglandin synthesis is not observed in in vitropreparations of this particular coral (2Brash A.R. Baertschi S.W. Ingram C.D. Harris T.M. J. Biol. Chem. 1987; 262: 15829-15839Abstract Full Text PDF PubMed Google Scholar). Using another species, the Arctic coral Gersemia fruticosa, a body of evidence indicates an endoperoxide pathway akin to the mammalian cyclooxygenase route (3Varvas K. Koljak R. Järving I. Pehk T. Samel N. Tetrahedron Lett. 1994; 35: 8267-8270Crossref Scopus (21) Google Scholar, 4Varvas K. Järving I. Koljak R. Valmsen K. Brash A.R. Samel N. J. Biol. Chem. 1999; 274: 9923-9929Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Although it has proved difficult to detect prostaglandin synthesis using in vitro preparations of P. homomalla, the metabolism of added arachidonic acid is readily observed (2Brash A.R. Baertschi S.W. Ingram C.D. Harris T.M. J. Biol. Chem. 1987; 262: 15829-15839Abstract Full Text PDF PubMed Google Scholar, 5Corey E.J. Matsuda S.P.T. Nagata R. Cleaver M.B. Tetrahedron Lett. 1988; 29: 2555-2558Crossref Scopus (60) Google Scholar). There is very high 8R-lipoxygenase activity, sufficient to consume perhaps 1 mm arachidonic acid in a 1:10 tissue/buffer homogenate. At least two distinct lipoxygenases account for this activity. One is a predominantly soluble enzyme, essentially a typical lipoxygenase in terms of catalytic properties, primary structure, and molecular weight (6Brash A.R. Boeglin W.E. Chang M.S. Shieh B.-H. J. Biol. Chem. 1996; 271: 20949-20957Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The second enzyme, the subject of the present investigation, is unique among reported lipoxygenases in that it occurs as a natural fusion protein with an extra 43-kDa N-terminal domain that functions as an allene oxide synthase (7Koljak R. Boutaud O. Shieh B.-H. Samel N. Brash A.R. Science. 1997; 277: 1994-1996Crossref PubMed Scopus (100) Google Scholar). Allene oxides are unstable epoxides formed from lipoxygenase-derived fatty acid hydroperoxides. Biosynthesis has been detected in both plants and marine invertebrates (8Hamberg M. J. Am. Oil Chem. Soc. 1989; 66: 1445-1449Crossref Scopus (26) Google Scholar, 9Song W.-C. Brash A.R. Bailey J.M. Prostaglandins, Leukotrienes, Lipoxins and PAF. Plenum Press, New York1991: 89-96Crossref Google Scholar). Allene oxides can cyclize to form five-membered carbon rings, and initially this was suspected to be part of the coral route to the prostaglandins (2Brash A.R. Baertschi S.W. Ingram C.D. Harris T.M. J. Biol. Chem. 1987; 262: 15829-15839Abstract Full Text PDF PubMed Google Scholar, 5Corey E.J. Matsuda S.P.T. Nagata R. Cleaver M.B. Tetrahedron Lett. 1988; 29: 2555-2558Crossref Scopus (60) Google Scholar). In plants, the enzymatic cyclization of an allene oxide derived from 13-hydroperoxylinolenic acid is established as a step in biosynthesis of the cyclopentanone hormone, jasmonic acid (10Hamberg M. Gardner H.W. Biochim. Biophys. Acta. 1992; 1165: 1-18Crossref PubMed Scopus (224) Google Scholar). Although the allene oxide pathway has not been substantiated for prostaglandin synthesis in coral, it may constitute the route to other prostanoid-related products such as the clavulones and punaglandins of other corals (5Corey E.J. Matsuda S.P.T. Nagata R. Cleaver M.B. Tetrahedron Lett. 1988; 29: 2555-2558Crossref Scopus (60) Google Scholar, 11Corey E.J. Lansbury P.T.J. Yamada Y. Tetrahedron Lett. 1985; 26: 4171-4174Crossref Scopus (44) Google Scholar, 12Baker B.J. Yu R.K. Okuda T.K. Scheuer P.J. J. Am. Chem. Soc. 1991; 107: 2976-2977Crossref Scopus (115) Google Scholar). There is also the implication of other as yet undefined roles for allene oxides. In starfish oocytes, for example, allene oxide synthesis occurs in the absence of enzymatic cyclization of the epoxide (13Brash A.R. Hughes M.A. Hawkins D.J. Boeglin W.E. Song W.-C. Meijer L. J. Biol. Chem. 1991; 266: 22926-22931Abstract Full Text PDF PubMed Google Scholar), and this also appears to be the case in P. homomalla (2Brash A.R. Baertschi S.W. Ingram C.D. Harris T.M. J. Biol. Chem. 1987; 262: 15829-15839Abstract Full Text PDF PubMed Google Scholar). The enzyme involved in allene oxide synthesis in plants is a member of the cytochrome P450 family of hemoproteins and is designated as CYP74A (14Song W.-C. Funk C.D. Brash A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8519-8523Crossref PubMed Scopus (272) Google Scholar). In contrast to the typical P450 monooxygenase, this allene oxide synthase (AOS)1 does not require molecular oxygen or reducing equivalents from NADPH. The ferric form of the hemoprotein converts the 13S-hydroperoxides of linoleic and linolenic acids to the corresponding allene oxides with a turnover number of ≈1000/s (15Song W.-C. Brash A.R. Science. 1991; 253: 781-784Crossref PubMed Scopus (232) Google Scholar). The plant AOS is closely related to the aldehyde-forming hydroperoxide lyase, another plant cytochrome P450 specialized for the metabolism of fatty acid hydroperoxides (16Matsui K. Shibutani M. Hase T. Kajiwara T. FEBS Lett. 1996; 394: 21-24Crossref PubMed Scopus (132) Google Scholar). The plant and coral AOS enzymes catalyze reactions with identical chemistry. It was unexpected, therefore, when cloning of the second lipoxygenase from P. homomalla led to characterization of a novel type of allene oxide synthase unrelated to the cytochrome P450 s (7Koljak R. Boutaud O. Shieh B.-H. Samel N. Brash A.R. Science. 1997; 277: 1994-1996Crossref PubMed Scopus (100) Google Scholar). The cDNA of the lipoxygenase-related transcript encoded a protein with a predicted molecular mass of 122 kDa. Sequencing revealed a C-terminal lipoxygenase domain of 79 kDa and a unique N-terminal domain of 43 kDa with some weak homology to catalase. Expression of the fusion protein and of the two separate domains established the 8R-lipoxygenase activity of the C-terminal 79 kDa and the AOS activity of the catalase-related N-terminal domain (7Koljak R. Boutaud O. Shieh B.-H. Samel N. Brash A.R. Science. 1997; 277: 1994-1996Crossref PubMed Scopus (100) Google Scholar). The lipoxygenase forms a specific 8R-hydroperoxy fatty the substrate of the AOS of the two domains of this unique fusion protein is acid was from and acid from and were by Chem. Scopus Google Scholar), and 8R-HPETE was using of P. A.R. Baertschi S.W. Ingram C.D. Harris T.M. J. Biol. Chem. 1987; 262: 15829-15839Abstract Full Text PDF PubMed Google Scholar). the AOS domain with a C-terminal the AOS domain was the with the encoded as with the the of the type the of the AOS domain the was at a The of the AOS was by the of encoded acid by a and with the two the for the was encoded as and not the enzyme and it when the is by a in in which case the of the be The 8R-lipoxygenase domain was at the and expressed with acids from the by and a with the two the of the type The two domains were expressed in using a expression by B.J. P. J. Expression PubMed Scopus Google Scholar). typical of a was out as 1 of was with a of in and at at for of this was used to 10 of and for the was with of and at for The were at for 10 in a with of mm at for 10 and in 10 of mm mm mm 1 and on for The were at for in 10 of and at were was added at a of 1 The were for s using a at a of The resulting were at for at AOS activity is present in the The was used for the lipoxygenase domain, that it was in the using 10 The allene oxide synthase was purified the of T. J.M. N. J. Biol. Chem. 1993; 26: Full Text PDF Google Scholar). The was on a with mm mm at The was with the and the were with mm mm mm The was with mm mm of were and for the AOS activity. The were mm using a the lipoxygenase was the The of the enzyme preparations was by and protein using was out to the using as a the of AOS the was and and the protein from the absorbance at using a from the protein by the The in the was by the D.J. PubMed Scopus Google Scholar). of enzyme was to with of and of was added and by under a The was in two and the was and of were added to the and the was The absorbance was at and and the was from the absorbance and using an of D.J. PubMed Scopus Google Scholar). a for the absorbance of The lipoxygenase domain was by protein or from the protein of on a gel in to the of a of and on the of enzymes and were out essentially as (2Brash A.R. Baertschi S.W. Ingram C.D. Harris T.M. J. Biol. Chem. 1987; 262: 15829-15839Abstract Full Text PDF PubMed Google A.R. Boeglin W.E. Chang M.S. Shieh B.-H. J. Biol. Chem. 1996; 271: 20949-20957Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, A.R. Song W.-C. 1996; PubMed Google using or substrate and in mm with of as under were used for in absorbance at for of products from fatty or were in the were using the and J. PubMed Scopus Google or using of the products the was to to of products from the of the for were than and the was with and to were in at under to The AOS domain was by the in absorbance at of the substrate hydroperoxides using a essentially as A.R. Song W.-C. 1996; PubMed Google Scholar). activity was using the in absorbance at were initially by using a or with of in the for a of the products and for and of in was used for of fatty acid hydroperoxides. was out using an and of acid for acids and or for were with and trimethylsilyl were to in of using of carbon and with for was added and the products with were out in the using a mass to a gas with a fused or were at and the was to at 10 or The of the AOS was on an in the at The was at a with a The AOS and 8R-HPETE were in mm The AOS was used at a of substrate concentrations from to at substrate concentrations were using a with the of AOS to the were to a the in absorbance at was for The at substrate was the of The were to a to the of the enzyme for substrate The were the substrate with and the and were by using the The the number of with of an enzyme was from the of formed enzyme with the of enzyme Scholar). were out using enzyme concentrations in of mm an of 8R-HPETE substrate The AOS was added at concentrations to The in absorbance at was the enzyme was completely and the absorbance of 8R-HPETE were using a of at P. 1987; PubMed Scopus Google Scholar). The primary of the lipoxygenase domain of the coral fusion protein shows an with mammalian lipoxygenases such that of the fusion protein with the of mammalian lipoxygenases. of the coral lipoxygenase acids was in and a at the Expression of the lipoxygenase domain coli (BL21 cells) active enzyme, with the activity in the of The lipoxygenase was using it be purified by chromatography on a The enzyme was in the mm it its activity at The domain maximum rates of reaction at reactions were at to the of substrate and the of the activity acid was in a with of at the and a absorbance at the lipoxygenase was There was from the part of the are as the of absorbance at The lipoxygenase domain some and unusual catalytic activity in mm with mm at an be to the in the of the two M. Biophys. 1993; PubMed Scopus Google Scholar). of cations the the two and an additional in catalytic activity a of was The of cations a maximum at a of the enzyme not or Although the enzyme was not mm on activity was by of concentrations of Although the enzymatic activity is in the of the substrate by the lipoxygenase at arachidonic acid concentrations was not The enzyme a turnover number of in mm and 10 mm activity as a of arachidonic acid acid at concentrations from to was in a with of mm and with or that the absorbance at was the lipoxygenase was added and the in absorbance was The of reaction was as of 8R-HPETE of In the absence of the lipoxygenase domain activity from the in The for enzyme in the was the of The was not catalytically active in the of this not be used to the of the enzyme under for metabolism of substrate. The enzyme also was inhibited in its catalytic activity by other and at the of reaction with arachidonic acid the of the active of the reaction also the catalytically active enzyme and, led to the conversion of substrate. The lipoxygenase domain a preference for or fatty The fatty linoleic and were not The substrate was as reaction by and 20.3ω6 The products of these fatty acids in of the AOS with were at in a at of reaction the products were with and by and acid was to 8R-HPETE and were to by of the The from an identical mass to J.M. Brash A.R. PubMed Scopus Google Scholar), and the enzymatic is The from and and the is the with 20.3ω6 a as and products designated as of products in and was not absorbance in the and a time on than which is the of the of was and by in the as the The mass a at and high mass at to the at the mass the two were and with at a A.R. Boeglin W.E. Y. Biophys. PubMed Scopus Google Scholar). The mass indicates a acid of the In with this the and of the to the of the arachidonic acid A.R. Boeglin W.E. Y. Biophys. PubMed Scopus Google Scholar). is as The a at for the and a time with and it was by as the J.M. Brash A.R. PubMed Scopus Google Scholar), and the is Expression of the fusion protein or of the lipoxygenase domain in coli that were in the or of In expression of the allene oxide synthase domain acids a soluble C-terminal in the a of the allene oxide synthase using chromatography on a (7Koljak R. Boutaud O. Shieh B.-H. Samel N. Brash A.R. Science. 1997; 277: 1994-1996Crossref PubMed Scopus (100) Google Scholar). Expression was high sufficient that the a distinct The coral AOS was active the and it a at At the of reaction of the coral allene oxide synthase was as ≈1400 turnovers/s with a of as by of the of its 8R-HPETE This turnover number is to that reported for the unrelated allene oxide synthase of in reaction with its 13-hydroperoxylinolenic acid (15Song W.-C. Brash A.R. Science. 1991; 253: 781-784Crossref PubMed Scopus (232) Google for the allene oxide synthase. The of the AOS was on a as under The at substrate was the of The were to a to the of the enzyme for substrate The were the substrate and the were by using the In contrast to the AOS reaction with 13-hydroperoxylinolenic the coral enzyme a in its of reaction s of reaction at the coral enzyme was completely The the of formed the AOS was for enzyme concentrations and the to a of This to the number of catalytic to an enzyme of the reaction of 8R-HPETE and the that the of with the reaction of the natural 8R-HPETE substrate. The an of reaction of than of the with Both enzyme under the the an absorbance of reaction with the an absorbance of with a of the not the of reaction with 8R-HPETE. of the for that other weak rates of reaction with the coral AOS and to of the with at or of the with 8R-HPETE, in the Using the in absorbance at to of the coral AOS reaction at enzyme concentrations than were used to rates of of 8R-HPETE of At concentrations of catalase used to its reaction with the enzyme in the absorbance of 8R-HPETE. At catalase a in absorbance of 8R-HPETE This very weak is to of as reported using substrate O. N. P. E.J. Chang M. Press, Scholar). in the two separate domains of homomalla fusion protein was to of and catalytic The coral lipoxygenase domain is to the fusion it be in with The AOS domain is to be catalase itself, and in contrast to the P450 enzymes with AOS activity. Both domains expressed well in were readily purified with the of the and activity for at The catalytic activity of the lipoxygenase domain is by the of Li+, or have a and, in concentrations of the of In the absence of added has on the catalytic activity. of are ineffective and it appears that the enzyme is not the may the of the enzyme, and also the or of substrate. this a natural of substrate for the coral lipoxygenase In its natural marine P. homomalla is with which among other and mm Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, L. P. P. M. Biol. Scholar). the of the in of is in the 8R-lipoxygenase The be as in typical plant and the of may be mm on Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google I. P. Biol. 1991; PubMed Scopus Google and perhaps in I. P. 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The gives of second and it the active enzyme reported among the mammalian lipoxygenases that have been purified T. Adv. Google Scholar, J. P. J. Biochim. Biophys. Acta. 1993; PubMed Scopus Google Scholar). the coral lipoxygenase domain has a mass of 79 typical for the enzyme has a mass of it is that the extra in the plant enzyme not the of its high catalytic activity. does the that it is a It is in activity in this case by a The high activity of the coral 8R-lipoxygenase makes it well suited for partnership with the AOS domain, the turnover of which are in the metabolism of 8R-HPETE. The that the catalase-related AOS enzyme can with to the plant AOS which are P450 (14Song W.-C. Funk C.D. Brash A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8519-8523Crossref PubMed Scopus (272) Google Scholar, W.-C. Brash A.R. Science. 1991; 253: 781-784Crossref PubMed Scopus (232) Google Scholar). 20.3ω6 was used as an and reaction of the coral lipoxygenase domain involved the synthesis of small of a in to the There were of a very small of the corresponding from arachidonic to or to be when on the as the 8R-HPETE The 20.3ω6 is a substrate than arachidonic and its in the active is also by of a small of Brash A.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of a bis-allylic has been reported among lipoxygenases with the lipoxygenase of the M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Both the and P. homomalla enzymes are and it to be is some in the biosynthesis of the that and on the It is also that the reaction occurs to a small in other lipoxygenases. The bis-allylic products under to the typical a A.R. Boeglin W.E. Y. Biophys. PubMed Scopus Google Scholar, M. 1992; Scopus Google Scholar). acid is used in typical fatty acid synthesis of bis-allylic products can be of the by the and AOS domains is of activity. The are in The lipoxygenase activity the for in using the absorbance at it was to the arachidonic acid substrate by enzyme to rates of reaction were observed when the were the other the reaction was at the enzyme high activity for 1 at the enzyme was completely on the of and lipoxygenases have the of reaction products with Biophys. Res. 1989; PubMed Scopus Google Scholar, Biophys. 1991; PubMed Scopus Google Scholar, K. M. T. S. T. Y. T. Biochim. Biophys. Acta. 1996; PubMed Scopus Google Scholar). reaction of the lipoxygenase domain of the coral fusion protein by in the it was that the absorbance the of a of There was of products in the of of the absorbance at of and of the of products and of of a type of It appears that the of enzyme activity may be related to such as of the enzyme than a of the AOS domain is related to The high of reaction to and the enzyme is 1 of a with fast was to of the reaction is observed among enzymes that or or epoxide lipoxygenase and cyclooxygenase the molecular of this has not been of the protein is a primary Thromboxane synthase and show and has been to a reaction J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, L. J. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). show that the two domains of the unusual fusion protein of P. homomalla have activities well suited for the conversion of arachidonic acid to the allene oxide. It to be established the two domains of the fusion protein and the fatty acid hydroperoxide is to the AOS It has been out that the of the AOS domain with of the lipoxygenase metabolism J. M. Biophys. Res. 1992; PubMed Scopus Google Scholar). This the of the lipoxygenase domain in the of a AOS It is also that the for the conversion of specific fatty acid hydroperoxides to allene oxides is completely for the plant AOS P450 or for the coral AOS the AOS domain has in to catalase yet catalytic activity is an additional of the that require an of the of acids in We and for with the and Boeglin for with the and mass We are to and at the for of the P.