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

Abstract

Lipoxygenases are enzymes that are found ubiquitously in higher animals and plants, but have only recently been identified in a number of bacteria. The genome of the diazotrophic unicellular cyanobacterium Cyanothece sp. harbors two genes with homology to lipoxygenases. Here we describe the isolation of one gene, formerly named csplox2. It was cloned, and the protein was expressed in Escherichia coli and purified. The purified enzyme belongs to the group of prokaryotic mini lipoxygenases, because it had a molecular mass of 65 kDa. Interestingly, it catalyzed the conversion of linoleic acid, the only endogenously found polyunsaturated fatty acid, primarily to the bisallylic hydroperoxide 11R-hydroperoxyoctadecadienoic acid. This product had previously only been described for the manganese lipoxygenase from the take all fungus, Gaeumannomyces graminis. By contrast, CspLOX2 was shown to be an iron lipoxygenase. In addition, CspLOX2 formed a mixture of typical conjugated lipoxygenase products, e.g. 9R-and 13S-hydroperoxide. The conversion of linoleic acid took place with a maximum reaction rate of 31 s ؊1 . Incubation of the enzyme with [(11S)-2 H]linoleic acid led to the formation of hydroperoxides that had lost the deuterium label, thus suggesting that CspLOX2 catalyzes antarafacial oxygenation as opposed to the mechanism of manganese lipoxygenase. CspLOX2 could also oxidize diarachidonylglycerophosphatidylcholine with similar specificity as the free fatty acid, indicating that binding of the substrate takes place with a "tail-first" orientation. We conclude that CspLOX2 is a novel iron mini-lipoxygenase that catalyzes the formation of bisallylic hydroperoxide as the major product.

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

“…Genomic DNA was isolated from approximately 50 mg of frozen cell material, as previously described ( 17 ). For the amplifi cation of the genomic DNA of the fusion protein of LOX and catalase-related hemoprotein ( 24 ), forward and reverse primers containing BamHI and Hin dIII restriction sites were designed (forward primer, 5 ′ -CGGATCCATGGATAGTCGTGATCCGCGCACTG-3 ′ and reverse primer, 5 ′ -CAAGCTTTTAGATATTGGTGCTCATCATA-3 ′ ).…”

“…PCC 8801, and named them CspLOX1 and CspLOX2. While CspLOX2 belongs to the group of prokaryotic mini-LOXs ( 17 ), CspLOX1 represents a rare group of LOX enzymes found in few bacteria with only very low sequence homology to all well-characterized LOXs. Here we show that despite this low sequence homology, CspLOX1 still catalyzes a typical LOX reaction.…”

“…This domain is, nonetheless, not essential for the catalytic activity because the so-called mini-LOXs, consisting only of the C-terminal domain, are enzymatically active. These mini-LOXs are naturally found in a number of cyanobacteria (17)(18)(19)(20), but are also generated by genetic removal or by limited proteolysis ( 16,21 ).…”

“…Absorbance at 234 nm was recorded for the detection of conjugated hydroxy fatty acids, respectively, during all chromatographic steps. Activity of CspLOX1 against PC esters was measured as previously described ( 17 ). Methyl esters were separated by RP-HPLC using a column as described above and with a solvent system of solvent A [methanol/water/acetic acid (75:25:0.1, v/v/v)] and solvent B [methanol/acetic acid (100:0.1, v/v)] using the following gradient: fl ow rate of 0.18 ml/min, 0 Ϫ 5 min, 100% A; 5 Ϫ 10 min from 100% A to 100% B and fl ow rate increase to 0.36 ml/min; 10 Ϫ 20 min 100% B; 20 Ϫ 25 min from 100% B to 100% A; 25 Ϫ 30 min 100% A. SP-HPLC analysis was carried out using a column as described above with a solvent system of n -hexane/2-propanol/trifl uoroacetic acid (100:1:0.02, v/v/v) at a fl ow rate of 0.1 ml/min and CP-HPLC analysis was carried out as in the case of free hydroxylated fatty acid.…”

Catalytic Convergence of Manganese and Iron Lipoxygenases by Replacement of a Single Amino Acid

“…LOXs abstract the bisallylic hydrogen from C-3 of the 1Z,4Z-pentadiene of unsaturated fatty acids followed by insertion of molecular oxygen at C-1, C-5, or occasionally C-3 with biosynthesis of hydroperoxy fatty acids (26,27); the lipoxidation of 18:2n-6 is outlined in Fig. 1A.…”