Structural basis for lipoxygenase specificity. Conversion of the human leukocyte 5-lipoxygenase to a 15-lipoxygenating enzyme species by site-directed mutagenesis

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9 ,11, 14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.     

Structural basis for the positional specificity of lipoxygenases

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of mammalian lipoxygenases but, unfortunately, the structural reasons for lipoxygenase specificity are not well understood. Although there are no direct structural data on lipoxygenase/substrate interaction, experiments with modified fatty acid substrates and mutagenesis studies suggest that for 12- and 15-lipoxygenases, arachidonic acid slides into the substrate-binding pocket with its methyl end ahead. For arachidonate 5- and/or 8-lipoxygenation two alternative models for the enzyme/substrate interaction have been developed: 1) The orientation-determined model and 2) the space-determined model. This review explores the experimental data available on the mechanistic reasons for lipoxygenase specificity and concludes that each of the above-mentioned hypotheses may be valid for arachidonate 5-lipoxygenation under certain circumstances.     

Oxygenation of phosphatidylcholine by human polymorphonuclear leukocyte 15-lipoxygenase

A cell-free human polymorphonuclear leukocyte preparation containing both 15- and 5-lipoxygenase activities was found to oxygenate phosphatidylcholine at carbon-15 of the arachidonic acid moiety. No oxygenation at carbon-5 was found. Under similar incubation conditions, soybean and rabbit reticulocyte 15-lipoxygenases also oxygenated phosphatidylcholine, whereas rat basophilic leukemia cell 5-lipoxygenase, rabbit platelet 12-lipoxygenase and rat liver cytochrome P-450 preparations did not. Our results suggest that the oxygenation of phospholipids may be a unique property of the 15-lipoxygenases.     

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.     

Applicability of the Triad Concept for the Positional Specificity of Mammalian Lipoxygenases

The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less space-filling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.     

Affinity labeling of the rabbit 12/15-lipoxygenase using azido derivatives of arachidonic acid

Lipoxygenases are lipid-peroxidizing enzymes, which have been implicated in the pathogenesis of important diseases. They consist of a single polypeptide chain, which is folded into a two-domain structure. The large catalytic domain contains the putative substrate-binding pocket and the catalytic non-heme iron. To identify structural elements of the rabbit 12/15-lipoxygenase that are involved in enzyme/substrate and/or enzyme/product interaction, we synthesized a set of radioactively labeled lipoxygenase substrates carrying a photoreactive azido group (17-azido-ETE, 18-azido-ETE, 19-azido-ETE) and used these compounds as affinity probes. After photoaffinity labeling, the enzyme was digested proteolytically and modified tryptic cleavage peptides were identified by a combination of radio-HPLC and mass spectral analysis. Following this strategy, we observed covalent linkage of a cleavage peptide that contained Ile593, which has previously been identified as the sequence determinant for the positional specificity. These data are consistent with the previous suggestion that this peptide lines the substrate-binding pocket. Surprisingly, we also observed strong labeling of cleavage peptides originating from the N-terminal beta-barrel domain, and our mass spectral data suggested covalent linkage of oxidized affinity probes. Taken together, these results confirm the previous conclusion that Ile593 and surrounding amino acids are constituents of the active site, but they also implicate the N-terminal beta-barrel in enzyme/substrate and/or enzyme/product interaction.     

15-Lipoxygenation of leukotriene A(4). Studies Of 12- and 15-lipoxygenase efficiency to catalyze lipoxin formation

The unstable epoxide leukotriene (LT) A(4) is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA(4) to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100000xg supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100000xg supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K(m) for LTA(4) (9-27 microM), as compared to the other lipoxygenases tested (58-83 microM). The recombinant human 12-lipoxygenase demonstrated the lowest K(m) value for LTA(4) (9 microM) whereas the porcine leukocyte 12-lipoxygenase had the highest V(max). The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA(4) and 6S-LXA(4) together with the all-trans LXA(4) and LXB(4) isomers were isolated. Production of LXB(4) was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate was considerably more efficient to inhibit conversion of LTA(4) to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC(50) 1 and 50 microM, respectively) in the human platelet cytosolic fraction.     

A simple method for the preparation of (5Z,8Z,11Z,14Z)-16-hydroxyeicosa-5,8,11,14-tetraenoic acid enantiomers and the corresponding 14,15-dehydro analogues: role of the 16-hydroxy group for the lipoxygenase reaction

(5Z,8Z,11Z,13E)-15-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) is not well oxygenated by arachidonate 15-lipoxygenases because of two structural reasons: (i) it contains a hydrophilic OH-group in close proximity to its methyl end and (ii) it lacks the bisallylic methylene at C(13). We synthesized racemic (5Z,8Z,11Z,14Z)-16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) which still contains the bisallylic C(13), separated the enantiomers reaching an optical purity of >99% and tested them as substrates for 5- and 15-lipoxygenases. Our synthetic pathway, which is based on stereospecific hydrogenation of a polyacetylenic precursor, yielded substantial amounts (30%) of 14,15-dehydro-16-HETE in addition to 16-HETE. When 16-HETE was tested as lipoxygenase substrate, we found that it is well oxygenated by the soybean 15-lipoxygenase and by the recombinant human 5-lipoxygenase. Analysis of the reaction products suggested an arachidonic acid-like alignment at the active site of the two enzymes. In contrast, the product pattern of 16-HETE methyl ester oxygenation by the soybean lipoxygenase (5-lipoxygenation) may be explained by an inverse head to tail substrate orientation.     

Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.     

Lipoxygenase-catalyzed oxygenation of arachidonylethanolamide,a cannabinoid receptor agonist

Various purified lipoxygenases were incubated with [¹⁴C]arachidonylethanolamide which is an endogenous ligand for cannabinoid receptors. When radioactive products were analyzed by thin-layer chromatography, porcine leukocyte 12-lipoxygenase and rabbit reticulocyte and soybean 15-lipoxygenases produced polar compounds at about the same reaction rates as that of oxygenation of free arachidonic acid. In contrast, the reaction of human platelet 12-lipoxygenase proceeded at a much lower rate, and porcine leukocyte 5-lipoxygenase was totally inactive. The result indicated that the lipoxygenases, which had been shown previously to be capable of oxygenating esterified polyunsaturated fatty acids, were also active with the arachidonylethanolamide. High-performance liquid chromatography, ultraviolet and mass spectrometry and nuclear magnetic resonance spectroscopy identified the major product by leukocyte 12-lipoxygenase as 12-hydroperoxy-5,8,10,14-eicosatetraenoylethanolamide and that by 15-lipoxygenases as 15-hydroperoxy-5,8,11,13-eicosatetraenoylethanolamide. The 15-hydroxy derivative inhibited electrically-evoked contraction of mouse vas deferens with an IC₅₀ of 0.63 μM as well as arachidonylethanolamide (0.17 μM), but the 12-hydroxy derivative was much less effective.     

Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase.

We have produced a model to define the linoleate-binding pocket of pea 9/13-lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron-coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13-lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null-effect of mutating pea 9/13-lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9-lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13-lipoxygenase and cucumber 13/9-lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9-lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate-binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate-binding to mammalian 5-, 12- and 15-lipoxygenases, would appear to be true also for linoleate-binding to plant 9- and 13-lipoxygenases.     

Structural characterization of the catalytic domain of the human 5-lipoxygenase enzyme

Leukotrienes are inflammatory mediators involved in several diseases. The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Little structural information is available regarding 5-lipoxygenase. In this study, we found that the primary structure of the catalytic domain of human 5-lipoxygenase is similar to that of the rabbit 15-lipoxygenase. This similarity allowed the development of a theoretical model of the tertiary structure of the 5-lipoxygenase catalytic domain, using the resolved structure of rabbit 15-lipoxygenase as a template. This model was used in conjunction with primary and secondary structural information to investigate putative nucleotide binding sites, a MAPKAP kinase 2 phosphorylation site, and a Src homology 3 binding site on the 5-lipoxygenase protein, further. Results indicate that the putative nucleotide binding sites are spatially distinct, with one on the -barrel domain and the other(s) on the catalytic domain. The MAPKAP kinase 2 phosphorylation site involves a four amino acid insertion in mammalian 5-lipoxygenases that significantly alters molecular structure. This target for post-translational modification is both common and unique to 5-lipoxygenases. The Src homology 3 binding site, found in all lipoxygenases, appears to lack the characteristic left-handed type II helix structure of known Src homology 3 binding sites. These results, which highlight the unique nature of the MAPKAP kinase site, underscore the utility of structural information in the analysis of protein function. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0076-y.Electronic Supplementary Material available.     

Structural basis for pH-dependent alterations of reaction specificity of vertebrate lipoxygenase isoforms

Lipoxygenases have been classified according to their specificity of fatty acid oxygenation and for several plant enzymes pH-dependent alterations in the product patterns have been reported. Assuming that the biological role of mammalian lipoxygenases is based on the formation of specific reaction products, pH-dependent alterations would impact enzymes' functionality. In this study we systematically investigated the pH-dependence of vertebrate lipoxygenases and observed a remarkable stability of the product pattern in the near physiological range for the wild-type enzyme species. Site-directed mutagenesis of selected amino acids and alterations in the substrate concentrations induced a more pronounced pH-dependence of the reaction specificity. For instance, for the V603H mutant of the human 15-lipoxygenase-2 8-lipoxygenation was dominant at acidic pH (65%) whereas 15-H(p)ETE was the major oxygenation product at pH 8. Similarly, the product pattern of the wild-type mouse 8-lipoxygenase was hardly altered in the near physiological pH range but H604F exchange induced strong pH-dependent alterations in the positional specificity. Taken together, our data suggest that the reaction specificities of wild-type vertebrate lipoxygenase isoforms are largely resistant towards pH alterations. However, we found that changes in the assay conditions (low substrate concentration) and introduction/removal of a critical histidine at the active site impact the pH-dependence of reaction specificity for some lipoxygenase isoforms.     

Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.     

Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2

Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue, 15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet there is no overlap in their positional specificities. In this study, we investigated the determinants of positional specificity using a random chimeragenesis approach in combination with site-directed mutagenesis. Exchange of the C-terminal one-third of the 8-LOX with the corresponding portion of 15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity. The critical region was narrowed down to a cluster of five amino acids by expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia coli. Finally, a pair of amino acids, Tyr(603) and His(604), was identified as the positional determinant by site-directed mutagenesis. Mutation of both of these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val, respectively) converted the positional specificity from 8S to 90% 15S without yielding any other by-products. Mutation of the corresponding residues in 15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for one amino acid substitution and 70% at C-8 for the double mutant. Based on the crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite the open coordination position of the catalytic iron in a likely site for substrate binding. The change from 8 to 15 specificity entails a switch in the head to tail binding of substrate. Enzymes that react with substrate "head first" (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these positions, whereas lipoxygenases that accept substrates "tail first" (12-LOX and 15-LOX) have an aliphatic residue with a glutamine or aspartate. Thus, this positional determinant of the 8-LOX and 15-LOX-2 may have significance for other lipoxygenases.     

Arachidonate 12-lipoxygenases with reference to their selective inhibitors

Lipoxygenase is a dioxygenase recognizing a 1-cis,4-cis-pentadiene of polyunsaturated fatty acids. The enzyme oxygenates various carbon atoms of arachidonic acid as a substrate and produces 5-, 8-, 12- or 15-hydroperoxyeicosatetraenoic acid with a conjugated diene chromophore. The enzyme is referred to as 5-, 8-, 12- or 15-lipoxygenase, respectively. Earlier we found two isoforms of 12-lipoxygenase, leukocyte- and platelet-type enzymes, which were distinguished by substrate specificity, catalytic activity, primary structure, gene intron size, and antigenicity. Recently, the epidermis-type enzyme was found as the third isoform. Attempts have been made to find isozyme-specific inhibitors of 12-lipoxygenase, and earlier we found hinokitiol, a tropolone, as a potent inhibitor selective for the platelet-type 12-lipoxygenase. More recently, we tested various catechins of tea leaves and found that (-)-gallocatechin gallate was a potent and selective inhibitor of human platelet 12-lipoxygenase with an IC50 of 0.14 microM. The compound was much less active with 12-lipoxygenase of leukocyte-type, 15-, 8-, and 5-lipoxygenases, and cyclooxygenases-1 and -2.     

Selective expression of 15-lipoxygenase activity by cultured human keratinocytes

Human keratinocytes isolated from neonatal skin express 15-lipoxygenase activity at a level far greater than that of any of the other pathways for lipoxygenation of arachidonic acid. The 10,000 x g supernatant of sonicates of 10(6) keratinocytes generates 15-hydroxy-eicosatetraenoic acid from 5 micrograms/ml of arachidonic acid at a mean maximum rate of 38 ng/30 min at 37 degrees C, that is similar to the activity of the 15-lipoxygenase of human airway epithelial cells and greater than that of endothelial cells and leukocytes. The unique mediators derived from the 15-lipoxygenation of arachidonic acid, that stimulate secretion and exert hyperalgesic effects, may achieve a concentration in skin sufficient to regulate local cellular and neural functions.     

Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes.

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions.     

Enantioselective substrate specificity of 15-lipoxygenase 1

15-Lipoxygenases are lipid-peroxidizing enzymes which have been implicated in the pathogenesis of various diseases, such as inflammation, atherosclerosis, and osteoporosis. Although the crystal structures for several lipoxygenase isoforms have been solved, there is little information on the substrate alignment at the active site and its impact on the catalytic mechanism. Investigating the oxygenation of specifically designed hydroxy fatty acids, we observed a pronounced enantioselectivity of 15-lipoxygenases for substrates carrying the oxygen moiety in close proximity to the site of hydrogen abstraction [16(R/S)-HETE, 17(R/S)-HETE]. To investigate the mechanistic basis for this unexpected behavior, we applied a strategy involving targeted substrate modification, site-directed mutagenesis, and structural modeling of the enzyme-substrate complex. Taken together, our data suggest that an (S)-hydroxy group in 16-HETE may form a hydrogen bridge between the substrate molecule and Gln548, which contributes to proper alignment of the fatty acid derivative at the active site of the enzyme. This interaction, which was not observed with 16(R)-HETE, 18(R)-HETE, or 18(S)-HETE, appears to be a major reason for the high degree of enantioselectivity during lipoxygenation of 16-HETE.     

The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding

The rabbit reticulocyte-type 15-lipoxygenase is capable of oxygenating biomembranes and lipoproteins without the preceding action of ester lipid cleaving enzymes. This reaction requires an efficient membrane binding, and the N-terminal beta-barrel domain of the enzyme has been implicated in this process. To obtain detailed information on the structural requirements for membrane oxygenation, we expressed the rabbit wild-type 15-lipoxygenase, its beta-barrel deletion mutant (catalytic domain), and several lipoxygenase point mutations as His-tagged fusion proteins in Escherichia coli and tested their membrane binding characteristics. We found that: (i) the beta-barrel deletion mutant was catalytically active and its enzymatic properties (K(M), V(max), pH optimum, substrate specificity) were similar to those of the wild-type enzyme; (ii) when compared with the wild-type lipoxygenase, the membrane binding properties of the N-terminal truncation mutant were impaired but not abolished, suggesting a role of the catalytic domain in membrane binding; and (iii) Phe-70 and Leu-71 (constituents of the beta-barrel domain) but also Trp-181, which is located in the catalytic domain, were identified as sequence determinants for membrane binding. Mutation of these amino acids to more polar residues (F70H, L71K, W181E) impaired the membrane binding capacity of the recombinant enzyme. These data indicate that the C-terminal catalytic domain of the rabbit 15-lipoxygenase is enzymatically active and that the membrane binding properties of the enzyme are determined by a concerted action of the N-terminal beta-barrel and the C-terminal catalytic domain.