X-ray crystallographic analysis and sequence comparisons of zinc enzymes have identified onsensus... more X-ray crystallographic analysis and sequence comparisons of zinc enzymes have identified onsensus sequences for catalytic and structural zinc binding sites (1). Such reference structures have served to predict zinc binding ligands among members of related zinc enzyme families and also, more noticeable, in apparently unrelated proteins. Thus, comparison of the amino acid sequence of LTA4 hydrolase with the corresponding primary structures of certain aminopeptidases and neutral proteases, typified by thermolysin, revealed the presence of a putative catalytic zinc site (1, 2). Consequently, LTA4 hydrolase was found, not only to contain one zinc atom per molecule, essential for the catalytic activity, but also to possess a zinc-dependent peptidase activity towards synthetic amides (3–5).
The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) ... more The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p3...
Proceedings of the National Academy of Sciences of the United States of America, Jan 11, 2016
Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the pot... more Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a cry...
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that possesses bo... more Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that possesses both an epoxide hydrolase activity, i.e., the well-known conversion of LTA4 into the proinflammatory substance LTB4, and a recently discovered peptidase activity. We have employed biochemical/kinetic analyses of native enzyme as well as site directed mutagenesis towards a recombinant enzyme to explore structural and functional properties of the enzyme active center. Thus, we have found that the peptidase activity is selectively stimulated by chloride ions, in a manner that suggests the presence of an anion binding site. Furthermore, a number of mutated enzymes have been constructed, expressed in E. coli, and purified to homogeneity to allow enzyme activity determinations and zinc analyses. The catalytic properties and zinc contents of these mutated enzymes establish the three zinc binding ligands of the protein and identify Glu-296 as a catalytic amino acid, directly involved in the peptid...
The Journal of pharmacology and experimental therapeutics, 1995
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes th... more Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of the unstable epoxide intermediate LTA4 into the proinflammatory substance LTB4 and also exhibits an amidase/peptidase activity toward synthetic substrates. Based on proposed reaction mechanisms for other zinc hydrolases, we have synthesized inhibitors of LTA4 hydrolase and evaluated their effects on the formation of LTB4 from LTA4 using both purified enzyme and intact polymorphonuclear leukocytes. The two most effective inhibitors, an alpha-keto-beta-amino ester (compound IV) and a thioamine (compound VIII), exhibited IC50 values of 1.9 +/- 0.9 and 0.19 +/- 0.12 microM (mean +/- SD, n = 4), respectively. Compounds IV and VIII were also potent inhibitors of LTB4 biosynthesis in ionophore stimulated polymorphonuclear leukocytes with IC50 < 200 nM. At higher concentrations, the biosynthesis of 5-hydroxy-eicosatetraenoic acid was also inhibited with IC50 approximately 10 m...
Advances in Experimental Medicine and Biology, 1997
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a soluble monomeric protein with a molecular mass o... more Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a soluble monomeric protein with a molecular mass of 69 kDa that converts the unstable epoxide intermediate LTA4 into the proinflammatory compound LTB4, 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosa-tetraenoic acid (For reviews see Refs. 1 and 2). Sequence comparisons of LTA4 hydrolase with certain zinc containing proteases and peptidases led to the discovery of a zinc binding motif in the primary structure of the enzyme3. Further studies verified that LTA4 hydrolase indeed contained one zinc atom per enzyme molecule4,5. These findings also led to the discovery of a previously unknown peptidase/amidase activity5,6 which was specifically stimulated by monovalent anions, e. g., chloride ions7, and also by albumin8. Studies involving site directed mutagenesis in combination with metal determination of the purified mutated proteins demonstrated that the zinc atom was bound to His-295, His-299, and Glu-318, in accordance with previous predictions, and that replacements of either of these three residues led to enzymatically inactive proteins which did not contain zinc9.
Proceedings of the National Academy of Sciences, 1992
The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zin... more The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis. Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes [Gln296]LTA4 hydrolase and [Ala296]LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme. In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be v...
Proceedings of the National Academy of Sciences, 1996
Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hy... more Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent ho...
Proceedings of the National Academy of Sciences, 1991
Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by sit... more Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coli, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained significant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA. Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed i...
Proceedings of the National Academy of Sciences, 2011
Lipoxygenases (LO) are a class of dioxygenases, which form hydroperoxy, hydroxy, and epoxy deriva... more Lipoxygenases (LO) are a class of dioxygenases, which form hydroperoxy, hydroxy, and epoxy derivatives of arachidonic acid with distinct positional and stereochemical configurations. In man, there are two known types of 12-LO that are distinguished by their expression patterns and catalytic properties. The platelet 12S-LO plays a role in platelet aggregation and 12R-LO seems to be important for normal skin function. Using BLAST searches of the zebrafish (zf) genome we identified one candidate zf12-LO gene with 43% identity with human 12R-LO at the mRNA level and the deduced primary sequence carried the so called “Coffa” structural determinant (Gly residue) for R stereoselectivity of LOs. However, incubations of recombinant, purified, zf12-LO with arachidonic acid revealed exclusive formation of 12( S )-hydroperoxy-eicosatetraenoic acid. Further studies with immunohistochemistry showed prominent expression of zf12-LO in the cell nuclei of skin epithelium, the epithelial lining of the...
Cyclooxygenase-2 (COX-2) is considered to play a major role in inflammation processes by catalyzi... more Cyclooxygenase-2 (COX-2) is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins (PGs). Using in situ hybridization histochemistry we studied the localization of COS-1 and COX-2 mRNA in the spinal cord and dorsal root ganglia (DRGs) after peripheral inflammation or after axotomy in the rat. No COX-2 mRNA signals were detected in the spinal cord under normal conditions, but strong expression was seen bilaterally in non-neuronal cells within the grey and white matter and along the leptomeninges and blood vessels 6 h after unilateral carrageenan injection into the hind paw, but not after peripheral nerve injury. The results suggest that COX-2 expressed in non-neuronal cells contributes to PG production in and around the spinal cord under peripheral inflammatory processes.
X-ray crystallographic analysis and sequence comparisons of zinc enzymes have identified onsensus... more X-ray crystallographic analysis and sequence comparisons of zinc enzymes have identified onsensus sequences for catalytic and structural zinc binding sites (1). Such reference structures have served to predict zinc binding ligands among members of related zinc enzyme families and also, more noticeable, in apparently unrelated proteins. Thus, comparison of the amino acid sequence of LTA4 hydrolase with the corresponding primary structures of certain aminopeptidases and neutral proteases, typified by thermolysin, revealed the presence of a putative catalytic zinc site (1, 2). Consequently, LTA4 hydrolase was found, not only to contain one zinc atom per molecule, essential for the catalytic activity, but also to possess a zinc-dependent peptidase activity towards synthetic amides (3–5).
The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) ... more The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p3...
Proceedings of the National Academy of Sciences of the United States of America, Jan 11, 2016
Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the pot... more Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a cry...
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that possesses bo... more Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that possesses both an epoxide hydrolase activity, i.e., the well-known conversion of LTA4 into the proinflammatory substance LTB4, and a recently discovered peptidase activity. We have employed biochemical/kinetic analyses of native enzyme as well as site directed mutagenesis towards a recombinant enzyme to explore structural and functional properties of the enzyme active center. Thus, we have found that the peptidase activity is selectively stimulated by chloride ions, in a manner that suggests the presence of an anion binding site. Furthermore, a number of mutated enzymes have been constructed, expressed in E. coli, and purified to homogeneity to allow enzyme activity determinations and zinc analyses. The catalytic properties and zinc contents of these mutated enzymes establish the three zinc binding ligands of the protein and identify Glu-296 as a catalytic amino acid, directly involved in the peptid...
The Journal of pharmacology and experimental therapeutics, 1995
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes th... more Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of the unstable epoxide intermediate LTA4 into the proinflammatory substance LTB4 and also exhibits an amidase/peptidase activity toward synthetic substrates. Based on proposed reaction mechanisms for other zinc hydrolases, we have synthesized inhibitors of LTA4 hydrolase and evaluated their effects on the formation of LTB4 from LTA4 using both purified enzyme and intact polymorphonuclear leukocytes. The two most effective inhibitors, an alpha-keto-beta-amino ester (compound IV) and a thioamine (compound VIII), exhibited IC50 values of 1.9 +/- 0.9 and 0.19 +/- 0.12 microM (mean +/- SD, n = 4), respectively. Compounds IV and VIII were also potent inhibitors of LTB4 biosynthesis in ionophore stimulated polymorphonuclear leukocytes with IC50 < 200 nM. At higher concentrations, the biosynthesis of 5-hydroxy-eicosatetraenoic acid was also inhibited with IC50 approximately 10 m...
Advances in Experimental Medicine and Biology, 1997
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a soluble monomeric protein with a molecular mass o... more Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a soluble monomeric protein with a molecular mass of 69 kDa that converts the unstable epoxide intermediate LTA4 into the proinflammatory compound LTB4, 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosa-tetraenoic acid (For reviews see Refs. 1 and 2). Sequence comparisons of LTA4 hydrolase with certain zinc containing proteases and peptidases led to the discovery of a zinc binding motif in the primary structure of the enzyme3. Further studies verified that LTA4 hydrolase indeed contained one zinc atom per enzyme molecule4,5. These findings also led to the discovery of a previously unknown peptidase/amidase activity5,6 which was specifically stimulated by monovalent anions, e. g., chloride ions7, and also by albumin8. Studies involving site directed mutagenesis in combination with metal determination of the purified mutated proteins demonstrated that the zinc atom was bound to His-295, His-299, and Glu-318, in accordance with previous predictions, and that replacements of either of these three residues led to enzymatically inactive proteins which did not contain zinc9.
Proceedings of the National Academy of Sciences, 1992
The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zin... more The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis. Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes [Gln296]LTA4 hydrolase and [Ala296]LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme. In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be v...
Proceedings of the National Academy of Sciences, 1996
Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hy... more Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent ho...
Proceedings of the National Academy of Sciences, 1991
Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by sit... more Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coli, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained significant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA. Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed i...
Proceedings of the National Academy of Sciences, 2011
Lipoxygenases (LO) are a class of dioxygenases, which form hydroperoxy, hydroxy, and epoxy deriva... more Lipoxygenases (LO) are a class of dioxygenases, which form hydroperoxy, hydroxy, and epoxy derivatives of arachidonic acid with distinct positional and stereochemical configurations. In man, there are two known types of 12-LO that are distinguished by their expression patterns and catalytic properties. The platelet 12S-LO plays a role in platelet aggregation and 12R-LO seems to be important for normal skin function. Using BLAST searches of the zebrafish (zf) genome we identified one candidate zf12-LO gene with 43% identity with human 12R-LO at the mRNA level and the deduced primary sequence carried the so called “Coffa” structural determinant (Gly residue) for R stereoselectivity of LOs. However, incubations of recombinant, purified, zf12-LO with arachidonic acid revealed exclusive formation of 12( S )-hydroperoxy-eicosatetraenoic acid. Further studies with immunohistochemistry showed prominent expression of zf12-LO in the cell nuclei of skin epithelium, the epithelial lining of the...
Cyclooxygenase-2 (COX-2) is considered to play a major role in inflammation processes by catalyzi... more Cyclooxygenase-2 (COX-2) is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins (PGs). Using in situ hybridization histochemistry we studied the localization of COS-1 and COX-2 mRNA in the spinal cord and dorsal root ganglia (DRGs) after peripheral inflammation or after axotomy in the rat. No COX-2 mRNA signals were detected in the spinal cord under normal conditions, but strong expression was seen bilaterally in non-neuronal cells within the grey and white matter and along the leptomeninges and blood vessels 6 h after unilateral carrageenan injection into the hind paw, but not after peripheral nerve injury. The results suggest that COX-2 expressed in non-neuronal cells contributes to PG production in and around the spinal cord under peripheral inflammatory processes.
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Papers by Jesper Haeggström