cDNA Cloning, Expression, and Mutagenesis Study of Liver-type Prostaglandin F Synthase

Abstract

F series prostaglandins (PG)1 are widely distributed in various organs of mammals and exhibit a variety of biological activities including constriction of pulmonary arteries (1, 2). PGF 2 is synthesized from PGE 2 , PGD 2 , or PGH 2 by PGE 9-ketoreductase, PGD 11-ketoreductase, or PGH 9,11-endoperoxide reductase, respectively. PGF synthase (EC 1.1.1.188) was purified from bovine lung by Watanabe et al. (3). It forms 9␣,11␤-PGF 2 (4) from PGD 2 (PGD 2 11-ketoreductase activity) and PGF 2␣ from PGH 2 (PGH 2 9,11-endoperoxide reductase activity) on the same molecule in the presence of NADPH (3, 4). This enzyme catalyzes the reduction of other carbonyl compounds including 9,10-phenanthrenequinone (PQ) as well as that of PGD 2 and PGH 2 but does not catalyze the reduction of PGE 2 . Although PGD 2 11-ketoreductase activity was competitively inhibited by PQ, PGH 2 9,11-endoperoxide reductase activity was not inhibited by PGD 2 or PQ (3). PGF synthase belongs to the aldo-keto reductase family. The bovine lung PGF synthase is a monomeric protein with a M r of 36,666 consisting of 323 amino acids, and its amino acid sequence shows high homology compared with that of other aldo-keto reductase family members (5). PGF synthase has two isozymes, one in the lung (3) and the other one in the liver (6), with different K m values for PGD 2 (120 and 10 M, respectively). The regulation by metals, the sensitivity to chloride ions, the inhibition by CuSO 4 and HgCl 2 , and the profile of immuno-precipitation with anti-bovine lung PGF synthase antibody are different between the two isozymes (6). Although Kuchinke et al. (7) isolated a clone (PGFS II) of PGF synthase from bovine liver and determined its amino acid sequence, the 99% similarity with the amino acid sequence of lung PGF synthase and the high K m value for PGD 2 of this recombinant PGFS II indicated that its cDNA was that of the lung-type enzyme even though it had been isolated from liver. Until now, the primary structure of the liver-type PGF synthase and the amino acids related to the affinity for PGD 2

Molecular Identification of Cytosolic Prostaglandin E2 Synthase That Is Functionally Coupled with Cyclooxygenase-1 in Immediate Prostaglandin E2Biosynthesis

“…The K m value of the recombinant cPGES/p23 purified from E. coli was calculated to be 14 M, which was comparable to the K m of other cytosolic terminal PG synthases, including lungtype (10 M) and liver-type (25 M) PGFSs (35) and hematopoietic PGD 2 synthase (500 M) (36), and the V max value (190 nmol/min/mg) was also similar to that of PGFSs (200 -400 nmol/min/mg) (35), although the in vitro assay conditions for each enzyme differ. It should be noted that the activity of bacterially expressed cPGES/p23 appeared to be approximately 1 order lower than that of the enzyme purified from rat brain or the recombinant enzyme expressed in HEK293 cells.…”

“…This property is reminiscent of that of lipocalin-type PGD 2 synthase, which not only plays a role in production of PGD 2 in the central nervous system (31) but also binds to several lipophilic ligands, such as biliverdin, bilirubin, and thyroid hormones (32), and functions as a retinoid transporter (33). PGF synthase is a member of the aldo-keto reductase superfamily, the enzymes belonging to which exhibit reductase activities toward various carbonyl compounds in addition to PGH 2 (34,35). Whether cPGES/p23 displays enzymatic activity toward lipophilic substances other than PGH 2 remains to be elucidated.…”

Role of Candidate Genes Regulating Uterine Prostaglandins Biosynthesis for Maternal Recognition of Pregnancy in Domestic Animals

“…Several PGFS have been identified; three were isolated in the bovine: lung type prostaglandin F synthase (PGFS1) [45], lung type PGFS found in liver (PGFS2) [46], and liver type PGFS, also called dihydrodiol dehydrogenase 3 (DDBX) [47,48]. Others were also identified, respectively, in human (AKR1C3) [49], sheep [50], Trypanosoma brucei [51], and recently in the porcine endometrium [52].…”

Structural and Mutational Analysis of Trypanosoma brucei Prostaglandin H2 Reductase Provides Insight into the Catalytic Mechanism of Aldo-ketoreductases

“…Most members of AKR described so far are known to lose their reductase activity toward AKR physiological substrates while they retain quinone reductase activity in the absence of the catalytic tyrosine (45,46,49,50). TbPGFS is also different from the human PGFS because this latter enzyme completely eliminates PGD 2 , PGH 2 , and 9,10-PQ reductase activities in the absence of the catalytic tyrosine (18).…”

“…PGF 2␣ synthase (EC 1.1.1.188) was first isolated from mammals (14), and the mammalian enzymes, which belong to the 1C subfamily of the AKRs (see Refs. 15 and 16 for AKR superfamily classification), catalyze the reduction of PGH 2 to PGF 2␣ and PGD 2 to 9␣,11␤-PGF 2 (a stereoisomer of PGF 2␣ ) (17), in addition to the oxidation of 9␣,11␤-PGF 2 to PGD 2 (18,19). In mammals, PGF 2␣ is a potent mediator of various physiological processes (20 -22) including regulation of vascular tone, constriction of uterine muscle (23) and pulmonary arter-ies (24,25), and induction of luteolysis during the estrous cycle and prior to parturition (26,27).…”