Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a ∼30 kDa N-terminal domain and a ∼60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a ∼60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of R-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 × 10 6 and 10.9 × 10 6 M -1 ‚s -1 , respectively), the activation energy of the reaction being 4.5 ( 0.5 and 8.3 ( 0.9 kJ‚mol -1 for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, R-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio-and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inihibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio-(patho)logical conditions.Lipoxygenases are a family of monomeric non-heme, nonsulfur iron dioxygenases which catalyze the conversion of unsaturated fatty acids into conjugated hydroperoxides. Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1). They have been also implicated in atherosclerosis (2), brain aging (3), HIV infection (4), and kidney disease (5, 6). In plants, lipoxygenases favor germination and participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress (7). Recently, they have been also shown to initiate the programmed death (apoptosis) of plant cells (8). Soybean lipoxygenase-1 (LOX-1) 1 is widely used as a prototype for studying the homologous family of lipoxygenases from tissues of different species, both in structural (9-13) and in kinetic (14-18) investigations. The amino acid sequence (19) and three-dimensional structure (9, 10) of LOX-1 have been determined. The overall 839 amino acid residues of LOX-1, with a molecular mass of 93 840 Da, show 2 domains. The first 146 N-terminal amino acid residues form an 8-stranded -barrel, and the remaining 693 residues of the C-terminal domain are organized into 23 helices and 8 -strands. Mammalian lipoxygenases do ...

SummaryThe present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease-associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease-associated strain a 9-residues insertion was found to be located close to the type I Cu-site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification, while the clinical isolatederived sAniA was not functional. Thus, the results obtained suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival.

Lipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membranebinding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò , A. (2001) Biochemistry 40, 6819 -6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1-3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0 -4.5 M). Both chemical-and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable.