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

Abstract

Biological transformations of polyunsaturated fatty acids often lead to chemically unstable products, such as the prostaglandin endoperoxides and leukotriene A 4 epoxide of mammalian biology and the allene epoxides of plants. Here, we report on the enzymatic production of a fatty acid containing a highly strained bicyclic four-carbon ring, a moiety known previously only as a model compound for mechanistic studies in chemistry. Starting from linolenic acid (C18.33), a dual function protein from the cyanobacterium Anabaena PCC 7120 forms 9R-hydroperoxy-C18.33 in a lipoxygenase domain, then a catalase-related domain converts the 9R-hydroperoxide to two unstable allylic epoxides. We isolated and identified the major product as 9R,10R-epoxy-11trans-C18.1 containing a bicyclo[1.1.0]butyl ring on carbons 13-16, and the minor product as 9R,10R-epoxy-11trans,13trans,15cis-C18.3, an epoxide of the leukotriene A type. Synthesis of both epoxides can be understood by initial transformation of the hydroperoxide to an epoxy allylic carbocation. Rearrangement to an intermediate bicyclobutonium ion followed by deprotonation gives the bicyclobutane fatty acid. This enzymatic reaction has no parallel in aqueous or organic solvent, where ring-opened cyclopropanes, cyclobutanes, and homoallyl products are formed. Given the capability shown here for enzymatic formation of the highly strained and unstable bicyclobutane, our findings suggest that other transformations involving carbocation rearrangement, in both chemistry and biology, should be examined for the production of the high energy bicyclobutanes.catalase ͉ carbocation ͉ epoxide ͉ leukotriene ͉ bicyclobutonium ion

Biosynthesis of a linoleic acid allylic epoxide: mechanistic comparison with its chemical synthesis and leukotriene A biosynthesis

“…Initially, we experimented with HPLC under cold conditions (silica column, hexane/diethyl ether solvent, 100:5, v/v, run at 210-15°C) as used previously for isolation of allene epoxides (17). Later, the use of HPLC conditions previously developed for chromatography of LTA 4 proved more satisfactory, either SP-HPLC of methyl esters using triethylamine in the solvent (14,18) or our modification of a reversed-phase HPLC (RP-HPLC) procedure, suitable for methyl esters or acids (1,19). For SP-HPLC, an Altex silica guard column (5 mm, 45 3 4.6 mm) was run with a solvent of hexane/triethylamine (100:0.5, by volume) eluted at 3 ml/min.…”

“…This is an unusual LOX activity, forming products enantiomeric to the 9S-LOX of plants (5), and characterized earlier in Hydra vulgaris (6), in the marine alga Ulva conglobata (7), and as an activity of Gersemia fruticosa arachidonate 11R-LOX (8). We showed that the catalaserelated domain converts the 9R-hydroperoxide of a-linolenic acid to two unstable epoxides that we isolated and characterized (1). One is an allylic 9,10-epoxide with C13-C16 of the carbon chain cyclized into a [1.1.0]bicyclobutane, a unique structural motif in biology.…”

“…strain PCC 7120 was cloned into pET17b for expression in E. coli as described previously (1). The full-length construct included the native cDNA sequence with a (His) 6 tag on the N terminus (MHHHHHHDLNTY--LMMSINI.).…”

“…One is an allylic 9,10-epoxide with C13-C16 of the carbon chain cyclized into a [1.1.0]bicyclobutane, a unique structural motif in biology. A second product, an allylic epoxide of the leukotriene A (LTA) type, 9R,10R-octadeca-11E,13E,15Z-trienoic acid, is also formed from the same 9R-hydroperoxylinolenic acid substrate (1).…”

A Bisallylic Mini-lipoxygenase from Cyanobacterium Cyanothece sp. That Has an Iron as Cofactor

“…These enzymes are significantly smaller than the described animal and plant LOXs and a number of them have so far been identified in cyanobacteria (35,(42)(43)(44)(45). Alignment of the predicted amino acid sequence of CspLOX2 with that of Mn-LOX shows conserved features, e.g.…”