| 1. |
- Hammarberg, Tove
(författare)
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5-Lipoxygenase : studies on the active site iron, and on the stimulatory factors Ca2+ and ATP
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human 5-lipoxygenase (5LO) is a monomeric non-heme iron enzyme, which catalyses the first two steps in the biosynthesis of leukotrienes: oxygenation of arachidonic acid to yield 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) and the subsequent dehydration to form the unstable epoxide intermediate 5(S)-trans-5,6-oxido7,9-trans-11,14-cis-eicosatetraenoic acid. The latter compound (leukotriene A4), is the precursor of the biologically active leukotrienes B4, C4, D4 and E4, which are regarded as critical mediators in asthma and other inflammatory and allergic disorders. The aim of the present study was to increase the understanding of 5-lipoxygenase catalysis and regulation. This thesis concerns the coordination of the active site iron in 5LO, and how the stimulatory factors ATP and Ca2+activate 5LO. Regarding the active site iron, the C-terminal isoleucine residue (I673) was shown to be essential for enzyme activity and for keeping the enzyme-bound iron. This indicated that 1673, in addition to two previously identified histidines (H372 and H550), constitutes a permanent and crucial ligand to the active site iron. Three other residues (H367, E376 and N554, conserved in most lipoxygenases) were important for catalytic activity, but were not needed to retain the active site iron. Further investigations using electron paramagnetic resonance and comparisons to other lipoxygenases, suggested that also H367 and N554 are iron ligands in 5LO, while E376 appeared to be part of the H-bond network which stabilises the iron centre. It seems possible that the bonds between the iron and H367 and/or N554 are weakened (possibly even broken) during certain states of the catalytic cycle, to enable direct interactions of other molecules (e.g. substrate) with the iron ion. Ca2+ and ATP were shown to stimulate 5LO independently. These factors were also demonstrated to bind directly and specifically to 5LO. Evidence for an unusual nucleotide-binding site in human 5LO was provided. This site seemed not to be located in the vicinity of the active site. Using ATP-analogues, two sequences which should be located close to or within the ATP-binding site in 5LO were identified. Ca2+ stimulation of 5LO, is regarded to be a key event in regulation of 5LO activity. The Ca2+ binding was localised to the N-terminal domain of 5LO, and molecular modelling supported that the N-terminal domain of 5LO constitutes a ß-barrel domain, which functions as a C2 domain in the Ca2+ regulation of 5LO activity. The requirement of micromolar levels of Ca2+ for membrane association of 5LO was confirmed. In addition, millimolar levels of Mg2+ could (at least to some extent) substitute for Ca2+ regarding both enzyme activity stimulation and the induced protein hydrophobicity enabling membrane association. These Mg2+ effects were also mediated by the N-terminal domain. The structural and functional similarities between 5LO and C2 domain proteins suggest that the 5LO N-terminal domain is responsible not only for the Ca2+ binding leading to membrane association, but also for the membrane interaction itself, and/or for protein-protein interactions.
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| 2. |
- Niegowski, Damian, 1978-, et al.
(författare)
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Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 448:7153, s. 613-616
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Tidskriftsartikel (refereegranskat)abstract
- Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase1: this eaction is the key step in cysteinyl leukotriene formation. Here we present the rystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A ̊resolution, respectively. The structure reveals a homotrimer, here each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a orseshoe-shaped conformation on GSH, and effectively positions the thiol group or activation by a nearby arginine at the membrane–enzyme interface. In addition, the structure provides a model for how the v-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular ‘ruler’ to align the eactive epoxide at the thiol of glutathione. This provides new structural insights nto the mechanism of LTC4 formation, and also suggests that the observed inding and activation of GSH might be common for a family of homologous proteins mportant for inflammatory and detoxification responses.
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