A 49-kDa Mini-lipoxygenase from Anabaena sp. PCC 7120 Retains Catalytically Complete Functionality

Abstract

Anabaena sp. PCC 7120 is one of the few prokaryotes harboring a lipoxygenase (LOX) gene. The sequence resides in an open reading frame encoding a fusion protein of a catalase-like hemoprotein with an unusually short LOX (ϳ49 kDa) at the C terminus. The recombinant mini-LOX contains a non-heme iron in the active site and is highly active with linoleic and ␣-linolenic acids (which occur naturally in Anabaena) giving the respective 9R-hydroperoxides, the mirror image of the 9S-LOX products of plants. Using stereospecifically labeled [11-3 H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C 16 /C 18:2 -phosphatidylcholine with 9R specificity, suggesting a "tail first" mode of fatty acid binding. Site-directed mutagenesis of an active site Ala (Ala 215 ), typically conserved as Gly in R-LOX, revealed that substitution with Gly retained 9R specificity, whereas the larger Val substitution switched oxygenation to 13S, implying that Ala 215 represents the functional equivalent of the Gly in other R-LOX. Metabolism studies using a synthetic fatty acid with extended double bond conjugation, 9E,11Z,14Z-20:36, showed that the mini-LOX can control oxygenation two positions further along the fatty acid carbon chain. We conclude that the mini-LOX, despite lacking the ␤-barrel domain and much additional sequence, is catalytically complete. Interestingly, animal and plant LOX, which undoubtedly share a common ancestor, are related in sequence only in the catalytic domain; it is possible that the prokaryotic LOX represents a common link and that the ␤-barrel domain was then acquired independently in the animal and plant kingdoms. Lipoxygenases (LOX)3 oxygenate polyunsaturated fatty acids to specific hydroperoxide derivatives (1, 2). The enzymes occur widely in the eukaryotic world, being ubiquitous in plants and fungi, common in primitive animals such as corals and hydra, and again ubiquitous in higher animals (3, 4). The lipoxygenase proteins are comprised of a polypeptide chain of 75-80 kDa in animals, 94 -104 kDa in plants, and harbor a single non-heme iron in the active site. As shown by x-ray crystallography, the animal and plant LOX enzymes share the same overall topology, with the extra ϳ20 -30 kDa in the plant LOX being accounted for by extra loops between the otherwise well conserved sequences (5-8). Lipoxygenases have a two-domain structure, an N-terminal C2-like or ␤-barrel domain, and the larger, mainly ␣-helical, catalytic domain that holds the iron in the active site. In the catalytic domain the LOX enzymes share five well conserved amino acid ligands to the iron, four histidines (or three His and one Asn or Ser) and the carboxylate group of the very C-terminal amino acid, usually Ile. Analysis of the structure-function of LOX enzymes suggests that the ␤-barrel domain plays a role in membrane binding and/or substrate acquisition (9, 10), whereas the catalytic domain is involved in controlling the reactio...

On the Substrate Binding of Linoleate 9‐Lipoxygenases

“…Additionally, we tested the activity of StLOX1 against a number of esterified fatty acids as substrates. Trilinolein and triarachidonin were used as substrates under the conditions described for the cucumber lipid body LOX, CslbLOX [5], while 1,2-diarachidonoyl-sn-glycero-3-phosphatidylcholine, as has been described for other LOX enzymes [8,9]. We could not observe activity against any of the substrates, which suggests that binding of the substrate takes place with the carboxyl-group first.…”

“…Based on recent mutagenesis data obtained for a LOX from Anabeana sp. PCC 7120, the existing model for stereo control of the LOX reaction may be expanded for enzymes that seem to have in general a bulkier amino acid in S-LOX at this position that controls stereospecificity [8,9].…”

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

“…The full-length fusion protein was successfully expressed in Escherichia coli, and although the cDNA encoding only the catalase-related domain did not express as active protein, the cDNA of the lipoxygenase (LOX) domain expressed very well by itself, allowing the dual catalytic activities of the fusion protein to be clearly distinguished. The LOX domain converts C18 fatty acids to the corresponding 9R-hydroperoxide (3,4). 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).…”

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

“…The same mode of binding has been described for 13S-LOXs (53, 54), 9R-LOXs (23,44), and Mn-LOX (51). On the other hand, a number of linoleate 9S-LOX and arachidonate 8S-and 5S-LOX cannot metabolize such substrates (55)(56)(57) and have been thought to bind fatty acids with the carboxylic end buried in the active site pocket.…”

“…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.…”