cDNA cloning, expression, and mutagenesis study of liver-type prostaglandin F synthase

Prostaglandin (PG) F synthase catalyzes the reduction of PGD(2) to 9 alpha,11 beta-PGF(2) and that of PGH(2) to PGF(2 alpha) on the same molecule. PGF synthase has at least two isoforms, the lung-type enzyme (K-m value of 120 mu M for PGD, (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O. (1985) J. Biol. Chem. 260, 7035-7041) and the liver-type one (K-m value of 10 mu M for PGD(2) (Chen, L. -Y., Watanabe, K., and Hayaishi, O. (1992) Arch. Biochem. Biophys. 296, 17-26)). The liver-type enzyme was presently found to consist of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with a M-r of 36,742. Sequence analysis indicated that the bovine liver PGF synthase had 87, 79, 77, and 76% identity with the bovine lung PGF synthase and human liver dihydrodiol dehydrogenase (DD) isozymes DD1, DD2, and DD4, respectively. Moreover, the amino acid sequence of the liver-type PGF synthase was identical with that of bo vine liver DD3. The liver-type PGF synthase was expressed in COS-7 cells, and its recombinant enzyme had almost the same properties as the native enzyme. Furthermore, to investigate the nature of catalysis and/or substrate binding of PGF synthase, we constructed and characterized various mutant enzymes as follows: R27E, R91Q, H170C, R223L, K225S, S301R, and N306Y. Although the reductase activities toward PGH, and phenanthrenequinone (PQ) of almost all mutants were not inactivated, the Ii, values of R27E, R91Q, H170C, R223L, and N306Y for PGD, were increased from 15 to 110, 145, 75, 180, and 100 mu M, respectively, indicating that Arg(27), Arg(91), His(170), Arg(223), and Asn(306) are essential to give a low Ii, value for PGD, of the liver-type PGF synthase and that these amino acid residues serve in the binding of PGD,. Moreover, the R223L mutant among these seven mutants especially has a profound effect on k(cat) for PGD(2) reduction. The K-m values of R223L, K225S, and S301R for PQ were about 2-10-fold lower than the wild-type value, indicating that the amino acid residues at 223, 225 and 301 serve in the binding of PQ to the enzyme. On the other hand, the K-m value of H170C for PGH(2) was 8-fold lower than that of the wild type, indicating that the amino acid residue at 170 is related to the binding of PGH(2) to the enzyme and that Cys(170) confer high affinity for PGH,. Additionally, the B-fold increase in k(cat)/K-m value of the N306Y mutant for PGH(2) compared with the wild-type value suggests that the amino acid at 306 plays an important role in catalytic efficiency for PGH(2).