Molecular Characterization of Prostamide/Prostaglandin F Synthase: A Novel Enzyme Belonging to the Thioredoxin Superfamily

Abstract

Purpose: : The purpose of these studies was to discover and characterize an enzyme that catalyzed to conversion of the prostamide H₂, the primary COX-2 derived oxidation product of anandamide, to prostamide F_2α

Methods: : _[¹⁴C] Prostamide H₂ was prepared from _[¹⁴C] anandamide using human COX-2, and was used as a substrate. _[¹⁴C] Prostamide H₂ assisted in the purification of prostamide / PGF synthase from swine brain and studies on the recombinant enzyme.The partial amino acid sequence of the highly purified enzyme was identified by Peptide Mass Fingerprint Analysis. (The molecular weights of the peptides digested by trypsin were determined by TOF-Mass). The amino acid sequences of these peptides were analyzed by using Mascot. Studies on the recombinant enzyme in E. coli, purification to homogeneity, and enzymatic assays for characterization and substrate specificity.

Results: : The recombinant and purified native enzyme catalysed the reduction of prostamide H₂ to prostamide F_2α with a Vmax of 0.25 µmol/min.mg and a Km of 7.6 µM. PGH₂ was similarly converted to PGF_2α. The mouse enzyme consisted of a 603-base pair open reading frame encoding a 201-amino acid protein with a molecular weight of 21,669. This enzyme belonged to the thioredoxin superfamily, which has the consensus region of _{⁴⁴Cys-x-x-⁴⁷Cys. Cys⁴⁴} represented the active site. Thioredoxin served as a reducing equivalent donor for the enzyme. In addition to enzymatic activity, Northerm and Western blots showed that the enzyme was predominately expressed in the CNS. Prostamide/PGF synthase activity was also detected in the porcine eye.

Conclusions: : A new enzyme that preferentially recognizes prostamide H₂ and PGH₂ was discovered and which belongs to the thioredoxin-like superfamily. This is the first report of a thioredoxin-based reduction system associated with the arachidonic acid cascade. It may have important functions in CNS and ocular tissue.

Keywords: eicosanoids • antioxidants • protein purification and characterization