5hv9
Human LTC4S mutant-S36E
Structural highlights
DiseaseLTC4S_HUMAN Defects in LTC4S are the cause of leukotriene C4 synthase deficiency (LTC4 synthase deficiency) [MIM:246530. LTC4 synthase deficiency is a fatal neurometabolic developmental disorder. It is associated with muscular hypotonia, psychomotor retardation, failure to thrive, and microcephaly. FunctionLTC4S_HUMAN Catalyzes the conjugation of leukotriene A4 with reduced glutathione to form leukotriene C4. Publication Abstract from PubMedLeukotriene C4 synthase (LTC4S) catalyzes the formation of the pro-inflammatory lipid mediator, leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes (cys-LTs), which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC mediated phosphorylation and recently, a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser-36 as the major p70S6k phosphorylation site, along with a low frequency site at Thr-40, using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation was tested with the phospho-mimetic mutant S36E, which displayed only about 20% (20 mumol/min/mg) of the activity of WT enzyme (95 mumol/min/mg), while the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state-kinetics analysis, indicating poor lipid substrate binding. The Ser-36 is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser-36 upon phosphorylation will pull the first luminal loop of LTC4S towards the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg-104 in the adjacent subunit. Since Arg-104 is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affects active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser-36 inhibits the catalytic function of LTC4S by interference with the catalytic machinery. Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity.,Ahmad S, Ytterberg AJ, Thulasingam M, Tholander F, Bergman T, Zubarev R, Wetterholm A, Rinaldo-Matthis A, Haeggstrom JZ J Biol Chem. 2016 Jun 30. pii: jbc.M116.735647. PMID:27365393[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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