Crystal structures of leukotriene A<sub>4</sub> hydrolase in complex with captopril and two competitive tight‐binding inhibitors

Thunnissen, Marjolein M.G.M.; Andersson, B. A.; Samuelsson, Bengt; Wong, Chi‐Huey; Haeggström, Jesper Z.

doi:10.1096/fj.01-1017fje

Cited by 69 publications

(67 citation statements)

References 48 publications

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“…This soluble enzyme converts LTA 4 into LTB 4 and thus provides an alternative fate for the product of the 5-LOX reaction. The crystal structure of LTA 4 hydrolase confirmed the prediction from sequence alignments that it is a member of the M1 amino peptidase superfamily (37,38). These are zinc-containing amino peptidases typified by thermolysin.…”

Section: Protein Structuressupporting

confidence: 68%

“…Consequently, regulation of pathway flux can be executed by the subcellular targeting of key enzymes. For example, as described below, targeting of 5-LOX to the outer nuclear membrane is thought to promote the formation of the cysteinyl-LTs, whereas 5-LOX targeting to the inner nuclear membrane results in the production of LTB 4 (37,38).…”

Section: Compartmentalizationmentioning

confidence: 99%

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Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

Newcomer

Gilbert

2010

Journal of Biological Chemistry

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Over the last 25؉ years, substantial progress has been made in understanding how LTs exert their effects, and a broader appreciation for the numerous biological processes they mediate has emerged. LT biosynthesis is initiated by the action of 5-lipoxygenase (5-LOX), which catalyzes the transformation of AA to LTA 4 in a two-step reaction. Ca 2؉ targets 5-LOX to the nuclear membrane, where it co-localizes with the 5-LOX-activating protein FLAP and, when present, the downstream enzyme LTC 4 synthase, both transmembrane proteins. Crystal structures of the AA-metabolizing LOXs, LTC 4 synthase, and FLAP combined with biochemical data provide a framework for understanding how subcellular organizations optimize the biosynthesis of these labile hydrophobic signaling compounds, which must navigate pathways that include both membrane and soluble enzymes. The insights these structures afford and the questions they engender are discussed in this minireview.The polyunsaturated fatty acid arachidonic acid (AA) 2 serves as a precursor to prostaglandins, leukotrienes (LTs), and other eicosanoids derived from the 20-carbon substrate. The proteins that constitute the LT biosynthetic pathway are located in several cellular compartments and include extra-and intracellular as well as membrane and soluble proteins. Products of the intracellular pathway are exported by specific pumps for further elaboration in extra-or transcellular biosynthesis or for access to the extracellular receptors they target. The complex compartmentalized biosynthetic pathway for LTs offers numerous opportunities for control of pathway flux through dynamic control of the locations of key proteins. We focus in this minireview on the early events in the LT biosynthetic pathway, specifically a major branch point in LT biosynthesis that determines whether cysteinyl-LTs that result from the pathway that requires conjugation with glutathione are produced or whether the non-cysteinyl-LT LTB 4 is synthesized. The Reaction PathwayThe biosynthesis of LTs is initiated by the action of 5-lipoxygenase (5-LOX), which promotes the transformation of AA to LTA 4 . The action of 5-LOX is triggered by calcium-dependent membrane binding, which targets it to the nuclear membrane. There it acquires its substrate, liberated from membrane phospholipids by the action of Ca 2ϩ -stimulated cytosolic phospholipase A 2 , from the transmembrane protein FLAP (five-lipoxygenase-activating protein). Like all LOXs, 5-LOX catalyzes the regio-and stereospecific peroxidation of a polyunsaturated fatty acid (1, 2). The enzyme transforms the substrate AA to the 5S-isomer of hydroperoxyeicosatetraenoic acid (5S-HPETE). However, in contrast to other members of this superfamily, 5-LOX can further metabolize the hydroperoxy product to an allylic epoxide, specifically LTA 4 . Studies have shown that both in vitro (3) and in vivo (4), the efficiency of LTA 4 production is improved with membrane binding, i.e. 5-LOX successfully carries the two-step reaction to completion, and the ratio of LTA 4 to inter...

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Section: Protein Structuressupporting

confidence: 68%

Section: Compartmentalizationmentioning

confidence: 99%

Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

Newcomer

Gilbert

2010

Journal of Biological Chemistry

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“…In keeping with these data is the model for binding of an arginyl tripeptide substrate to LTA4H. In this model, Tyr378, the residue corresponding to Phe544 in IRAP, plays a role in defining the S1 and S2Ј subsites of the active site (Thunnissen et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Phenylalanine-544 Plays a Key Role in Substrate and Inhibitor Binding by Providing a Hydrophobic Packing Point at the Active Site of Insulin-Regulated Aminopeptidase

Albiston¹,

Pham²,

Ye³

et al. 2010

Mol Pharmacol

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Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and sitedirected mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn 2ϩ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn 2ϩ ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the K i in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

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“…One patch of the cavity is hydrophilic, with Gln-134, Asp-375, and the hydroxyl of Tyr-267 clustering together. This cavity was probed by structural determination of complexes between LTA4H and specific, active site-directed inhibitors, some of which have been designed as LTA 4 mimics (35). Indeed, the hydrophobic tail of the inhibitors, corresponding to the fatty acid backbone of LTA 4 , is buried into the narrow hydrophobic pocket, strongly indicating that it functions as a substrate binding cavity (Fig.…”

Section: Crystal Structure Of Lta 4 Hydrolasementioning

confidence: 99%

Leukotriene A4 Hydrolase/Aminopeptidase, the Gatekeeper of Chemotactic Leukotriene B4 Biosynthesis

Haeggström

2004

Journal of Biological Chemistry

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171

181

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The leukotrienes (LTs) 1 are a family of lipid mediators that play important roles in a variety of allergic and inflammatory reactions (1, 2). These molecules are formed by leukocytes and are divided into two classes, the spasmogenic cysteinyl leukotrienes and LTB 4 , which is a classical chemoattractant that triggers adherence and aggregation of leukocytes to the endothelium at nanomolar concentrations. Recent data also indicate that LTB 4 is a chemoattractant for T-cells, creating a functional link between early innate and late adaptive immune responses to inflammation (3-5). In addition, LTB 4 participates in the host defense against infections (6) and is a key mediator of platelet-activating factor-induced lethal shock (7). Because of these powerful biological effects, LTB 4 is regarded as an important chemical mediator in a variety of acute and chronic inflammatory diseases, e.g. nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease (8). Moreover, only recently, several lines of pharmacological, morphological, biochemical, and genetic evidence have been gathered implicating LTs, in particular LTB 4 , as a mediator of vascular inflammation and arteriosclerosis (9). This article gives an overview of the biochemical, structural, and catalytic properties of LTA 4 hydrolase (LTA4H), which catalyzes the final and committed step in LTB 4 biosynthesis. LTA 4 Hydrolase Is a Key Enzyme in the 5-Lipoxygenase PathwayIn cellular biosynthesis of LTs, 5-lipoxygenase, assisted by 5-lipoxygenase-activating protein, converts arachidonic acid into the unstable epoxide LTA 4 , which in turn may be enzymatically conjugated with GSH to form LTC 4 , the parent compound of the cysteinyl leukotrienes, or hydrolyzed into LTB 4 by LTA4H (Fig. 1). Leukotrienes can also be formed via transcellular routes, where LTA 4 is donated from an activated leukocyte to a recipient cell for further metabolism by downstream enzymes, a process that was recently shown to occur in vivo (10). LTB 4 signals via a specific, high affinity, G-protein-coupled receptor (BLT 1 ) (11). In addition, a second receptor for LTB 4 (BLT 2 ) has been discovered, the functional role of which is presently not known (12). Interestingly, LTB 4 is also a natural ligand of the peroxisome proliferator-activated receptor ␣ class of nuclear receptors and has been suggested to play a role in lipid homeostasis (13). Leukotriene A 4 Hydrolase Is a Zinc-dependent Epoxide Hydrolase and AminopeptidaseLTA4H is a monomeric soluble protein that is widely distributed and has been purified from several mammalian sources (14). It resides in the cytosol, although nuclear localization has also been reported recently (15). The cDNAs encoding the human, mouse, rat, and guinea pig enzymes have been cloned and sequenced. The proteins contain 610 amino acids, excluding the first Met, and the calculated molecular mass of the human enzyme is 69,153 Da. The primary, secondary, and tertiary structures of LTA4H bear no resemblance to soluble xenobiotic epoxide hydrolase, alth...

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Crystal structures of leukotriene A₄ hydrolase in complex with captopril and two competitive tight‐binding inhibitors

Cited by 69 publications

References 48 publications

Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

Phenylalanine-544 Plays a Key Role in Substrate and Inhibitor Binding by Providing a Hydrophobic Packing Point at the Active Site of Insulin-Regulated Aminopeptidase

Leukotriene A4 Hydrolase/Aminopeptidase, the Gatekeeper of Chemotactic Leukotriene B4 Biosynthesis

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Crystal structures of leukotriene A4 hydrolase in complex with captopril and two competitive tight‐binding inhibitors

Cited by 69 publications

References 48 publications

Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

Phenylalanine-544 Plays a Key Role in Substrate and Inhibitor Binding by Providing a Hydrophobic Packing Point at the Active Site of Insulin-Regulated Aminopeptidase

Leukotriene A4 Hydrolase/Aminopeptidase, the Gatekeeper of Chemotactic Leukotriene B4 Biosynthesis

Contact Info

Product

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Crystal structures of leukotriene A₄ hydrolase in complex with captopril and two competitive tight‐binding inhibitors