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Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.
Arch Biochem Biophys. 2003 Nov 15; 419(2):139-46.AB

Abstract

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.

Authors+Show Affiliations

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14592457

Citation

Cho, Hoon, et al. "Critical Residues for the Coenzyme Specificity of NAD+-dependent 15-hydroxyprostaglandin Dehydrogenase." Archives of Biochemistry and Biophysics, vol. 419, no. 2, 2003, pp. 139-46.
Cho H, Oliveira MA, Tai HH. Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. Arch Biochem Biophys. 2003;419(2):139-46.
Cho, H., Oliveira, M. A., & Tai, H. H. (2003). Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. Archives of Biochemistry and Biophysics, 419(2), 139-46.
Cho H, Oliveira MA, Tai HH. Critical Residues for the Coenzyme Specificity of NAD+-dependent 15-hydroxyprostaglandin Dehydrogenase. Arch Biochem Biophys. 2003 Nov 15;419(2):139-46. PubMed PMID: 14592457.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. AU - Cho,Hoon, AU - Oliveira,Marcos A, AU - Tai,Hsin-Hsiung, PY - 2003/11/1/pubmed PY - 2004/1/24/medline PY - 2003/11/1/entrez SP - 139 EP - 46 JF - Archives of biochemistry and biophysics JO - Arch. Biochem. Biophys. VL - 419 IS - 2 N2 - 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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/14592457/Critical_residues_for_the_coenzyme_specificity_of_NAD+_dependent_15_hydroxyprostaglandin_dehydrogenase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003986103004752 DB - PRIME DP - Unbound Medicine ER -