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 the 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 Km and Vmax 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.
Ensor
C M
CM
Division of Medicinal Chemistry and Pharmaceutics, University of Kentucky College of Pharmacy, Lexington 40536-0082.
Tai
H H
HH
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology*; D010920:Placenta / Q000201:enzymology*; D011453:Prostaglandins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics*
D011453:Prostaglandins; D011994:Recombinant Proteins; D009243:NAD; D014443:Tyrosine; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D008239:Lysine