Purification and structural characterization of placental NAD+-linked 15-hydroxyprostaglandin dehydrogenase. The primary structure reveals the enzyme to belong to the short-chain alcohol dehydrogenase family

Abstract

Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, with chromatography on DEAE-Sepharose, Blue Sepharose, and Mono-Q FPLC as principal steps. Final yield was 23% and purification about 13,000-fold, with a specific activity of 24,000 milliunits/mg. The subunit molecular weight is about 29,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the native protein molecular weight is about 54,000 as estimated by Sephadex G-100 chromatography, establishing the enzyme to be a dimer of similar-sized protein chains. The subunit N-terminal residue is methionine, and the alpha-amino group is free. The complete primary structure was determined by peptide analysis, based essentially on four different proteolytic treatments (Lys-specific protease, Glu-specific protease, Asp-specific protease, and CNBr). The protein chain is composed of 266 residues, with C-terminal glutamine. A microheterogeneity was detected at position 217, with both Cys and Tyr, in about equal amounts, from a preparation starting with a single placenta. No other subunit heterogeneities were detected. The protein is clearly but distantly related to insect alcohol dehydrogenases, characterized bacterial dehydrogenases of sugar metabolism, and bacterial and eukaryotic steroid dehydrogenases. Together, these results establish that placental 15-hydroxyprostaglandin dehydrogenase is a member of the short-chain nonmetalloenzyme alcohol dehydrogenase protein family. The protein has four cysteine residues (five with the positional microheterogeneity), but there is no evidence for functional importance of any of these residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type

“…Divergent members of the 'short-chain dehydrogenase type' have been characterized and additional sugar, steroid and other dehydrogenases were added to the family [2,[4][5][6][7], as well as species variants for the Drosophila alcohol dehydrogenase [8]. The enzymes initially characterized were non-mammalian, but recently mammalian proteins have also been found to belong to this group, thus linking a human prostaglandin [9] and a human steroid dehydrogenase [10,11] to the prokaryotic/insect short-chain dehydrogenase type. Furthermore, a less variable segment and a strictly conserved Tyr residue have been defined [9], opening the possibility for mechanistic studies of this enzyme group, for which structure-function relationships have been little studied thus far.…”

“…The enzymes initially characterized were non-mammalian, but recently mammalian proteins have also been found to belong to this group, thus linking a human prostaglandin [9] and a human steroid dehydrogenase [10,11] to the prokaryotic/insect short-chain dehydrogenase type. Furthermore, a less variable segment and a strictly conserved Tyr residue have been defined [9], opening the possibility for mechanistic studies of this enzyme group, for which structure-function relationships have been little studied thus far. Consequently, additional …”

The Prostate Expression Database (PEDB)

“…Of relevance for the study of androgen-mediated effects in the prostate gland and other androgen-responsive tissues is the classification of several key enzymes involved in steroid hormone biosynthesis, hydroxysteroid dehydrogenases (HSDs), within the SDR family. This group of HSDs includes 17P-HSD types 1^ and 6-8 (Peltoketo et al, 1999), 15-hydroxyprostaglandin dehydrogenase (Krook et al, 1990), and lip-HSD (Oppermann et al, 1997b). 173-HSD is responsible for the conversion of androstenedione to its more potent form, testosterone (Norman and Litwack, 1997; Zhou and Speiser, 1999; Couture et al, 1993).…”

Three‐dimensional model of NAD+‐dependent 15‐hydroxyprostaglandin dehydrogenase and relationships to the NADP+‐dependent enzyme (carbonyl reductase)

“…The sequences of human placental NAD'-linked 15-hydroxyprostaglandin dehydrogenase [5] and 3cr/20fi-hydroxysteroid dehydrogenase from Streptomyces hydrogenans [IO] were aligned using the program ALIGN [1 1] to maximize sequence identity between the two proteins. The alignment was then adjusted manually in order to conserve secondary structure.…”

“…Two different cytosolic types of human prostaglandin dehydrogenase have been characterized, a monomeric NADP'-linked enzyme identical to carbonyl reductase [l-4], and a dimeric NAD'-linked enzyme [5]. Both belong to the short-chain dehydrogenase family, but they are highly divergent, exhibiting identical residues at the 20% level [1, 2,6].…”