Bacterial expression and site-directed mutagenesis of two critical residues (tyrosine-151 and lysine-155) of human placental NAD⁺ -dependent 15-hydroxyprostaglandin dehydrogenase

NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. In this study the cDNA for human placental 15-PGDH was expressed in Escherichia coli and che recombinant enzyme was purified to homogeneity and characterized. The N-terminus of the recombinant protein was sequenced and found to be identical with the known amino-acid sequence of 15-PGDH. Determinations of K_m and V_ax values for a number of the prostaglandins and NAD⁺ indicate that the recombinant enzyme does not appear to be kinetically different from the human placental enzyme. Site-directed mutagenesis was used to examine the importance of two residues which are highly conserved in the short-chain dehydrogenases which are known to be related to 15-PGDH. Tyrosine-151 was changed to phenylalanine and serine while lysine-155 was changed to glutamine and leucine. Western blot analysis indicated that the mutant and wild-type proteins were 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.