BACTERIAL EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF 2 CRITICAL RESIDUES (TYROSINE-151 AND LYSINE-155) OF HUMAN PLACENTAL NAD(-DEPENDENT 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE())

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catal yzes the first step in the catabolic pathway of the prostaglandins. Th is enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. In this s tudy the cDNA for human placental 15-PGDH was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity and chara cterized. The N-terminus of the recombinant protein was sequenced and found to be identical with the known amino-acid sequence of 15-PGDH. D eterminations of K-m and V-max values for a number of the prostaglandi ns and NAD(+) indicate that the recombinant enzyme does not appear to be kinetically different from the human placental enzyme. Site-directe d mutagenesis was used to examine the importance of two residues which are highly conserved in the short-chain dehydrogenases which are know n to be related to 15-PGDH. Tyrosine-151 was changed to phenylalanine and serine while lysine-155 was changed to glutamine and leucine. West ern blot analysis indicated that the mutant and wild-type proteins wer e expressed at the similar levels. However, all of the mutant proteins were found to be inactive. These results indicate that both tyrosine- 151 and lysine-155 are required for 15-PGDH activity.