Structure-function analysis of human l-prostaglandin D synthase bound with fatty acid molecules.
Human prostaglandin D synthase (L-PGDS) is a lipocalin-type enzyme involved in the metabolism of arachidonic acid and plays a key role in the regulation of sleep, allergy, pain sensation, and the development of male reproductive organs. Here, using a combination of crystallographic, biochemical, mutagenesis, and kinetic studies, we have gained insights into the mode of ligand binding by human L-PGDS and have identified residues involved in catalysis. Interestingly, structural evidence reveals that 2 molecules of fatty acids, one molecule each of oleic and palmitoleic acid, bind inside the β barrel. The oleic acid is buried and binds in a highly basic patch in proximity to the catalytically critical Cys65, mimicking the binding of prostaglandin H(2). The palmitoleic acid sits in a relatively neutral region with very few interactions with the protein. Mutating Met64, Leu79, Phe83, or Leu131 to alanine reduced the catalytic efficiency by almost 10-fold, while K59A and Y149A mutations enhanced the catalytic efficiency by >2-fold. Met64 seems to function as a kinetic clamp, pushing the thiol group of Cys65 close to the site of nucleophilic attack during catalysis.
Zhou
Yangyan
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Shaw
Neil
N
Li
Yang
Y
Zhao
Yu
Y
Zhang
Rongguang
R
Liu
Zhi-Jie
ZJ
D018360:Crystallography, X-Ray; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054834:Lipocalins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship
D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; D054834:Lipocalins; C008757:palmitoleic acid; D019301:Oleic Acid; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase