Structural and functional significance of cysteine residues of glutathione-independent prostaglandin D synthase. Identification of Cys65 as an essential thiol.
Glutathione-independent prostaglandin D synthase in rat brain is composed of 189 amino acid residues and catalyzes the isomerization of prostaglandin H2 to prostaglandin D2, an endogenous sleep-promoting substance. This enzyme is the only enzyme among members of the lipocalin superfamily composed of various secretory lipophilic ligand-carrier proteins and is recently identified to be a beta-trace protein, a major constituent of human cerebrospinal fluid. We expressed the active enzyme in Escherichia coli and then systematically substituted all cysteine residues of the delta 1-29 enzyme at positions of 65, 89, and 186 with alanine or serine. The parent and mutant enzymes were purified to apparent homogeneity with a recovery of approximately 30% by chromatography with Sephadex G-50 and S-Sepharose, by which all the enzymes showed identical elution profiles. The purified enzymes, irrespective of the mutation, showed almost the same circular dichroism spectral characteristics as displayed by a highly ordered beta-structure. The recombinant enzymes containing Cys65 showed the activity comparable with that of the enzyme purified from rat brain (approximately 3 mumol/min/mg of protein) in the presence, but not in the absence, of sulfhydryl compounds. However, all of the single, double, and triple mutants without Cys65 lost the enzyme activity. The purified delta 1-29 Ala89,186 enzyme was inactivated reversibly by conjugation with glutathione at Cys65 and irreversibly by the stoichiometric chemical modification with N-ethylmaleimide. These results indicate that Cys65 is an essential thiol of the enzyme and that both the intrinsic and extrinsic sulfhydryl groups are necessary for nonoxidative rearrangement of 9,11-endoperoxide of prostaglandin H2 to produce prostaglandin D2 catalyzed by the enzyme.
Urade
Y
Y
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Japan.
Tanaka
T
T
Eguchi
N
N
Kikuchi
M
M
Kimura
H
H
Toh
H
H
Hayaishi
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054834:Lipocalins; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds
D054834:Lipocalins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D005978:Glutathione; D003545:Cysteine