Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.
NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.
Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.
Hoon
H
Cho
Marcos A
MA
Oliveira
Hsin-Hsiung
HH
Tai
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003198:Computer Simulation; D017124:Conserved Sequence; D004789:Enzyme Activation; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D011453:Prostaglandins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003067:Coenzymes; D011453:Prostaglandins; D011993:Recombinant Fusion Proteins; D009243:NAD; D009249:NADP; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase