Threonine 11 of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase may interact with NAD(+) during catalysis.
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase family, 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. A relatively conserved threonine residue corresponding to threonine 11 of 15-PGDH is proposed to be involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine the important role of this residue. Threonine 11 was changed to alanine (T11A), cysteine (T11C), serine (T11S) or tyrosine (T11Y) and the mutant proteins were expressed in E. coli. Western-blot analysis showed that the expression levels of mutant proteins were comparable to that of the wild-type enzyme. Mutants T11A, T11C and T11Y were found to be inactive. Mutant T11S still retained substantial activity and the K(m) value for prostaglandin E(2) (PGE(2)) was similar to the wild-type enzyme; however, the K(m) value for NAD(+) was increased over 23-fold. These results suggest that threonine 11 may be involved in the interaction with NAD(+) either directly or indirectly and contributes to the full catalytic activity of 15-PGDH.
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Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington 40536-0082, USA.
Tai
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D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D017124:Conserved Sequence; D015232:Dinoprostone / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005470:Fluorometry; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D009243:NAD; D013912:Threonine; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D015232:Dinoprostone