Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases

Abstract

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins.

Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui

“…Determination of whether the putative bacterial or archaeal sequences of clusters IA and IB represent functional oxidoreductases or (xylose) dehydrogenases must await their biochemical characterization following expression of the genes that encode them. Eukaryotic xylose dehydrogenases-dimeric DDs, (cluster II), which probably represent a novel family of dehydrogenases (1,2), and archaeal glucose dehydrogenases (cluster III), which belong to the medium-chain dehydrogenase-reductase superfamily (26), form distinct phylogenetic clusters separate from the xylose dehydrogenase from H. marismortui, which is in accordance with differences in their molecular and catalytic properties.…”

Roles of His-79 and Tyr-180 of -Xylose/Dihydrodiol Dehydrogenase in Catalytic Function

“…The recombinant proteins were overexpressed in Escherichia coli BL21 (DE3), and the resulting proteins were purified as described in detail elsewhere (6,10). Since the Y180F mutant had extremely low activity under standard assay conditions, the eluants from the chromatography columns were monitored by SDS-PAGE (16) on 12.5% gels.…”

“…A recent report on the substrate specificity has shown that dimeric DD efficiently oxidizes several pentoses and is identical with NADP ϩ -dependent D-xylose dehydrogenase (EC 1.1.1.179) (9). The cloning of cDNAs for dimeric DDs of human and the above four animals (10) revealed that the enzymes of these species with 82-94% sequence identities show no homology with the monomeric DDs and cis-dihydrodiol dehydrogenases, which belong to the aldo-keto reductase family (11) and short-chain dehydrogenase/reductase family (12), respectively. However, dimeric DD shows sequence similarity to putative gene products of microorganisms and Zymomonas mobilis glucose-fructose oxidoreductase (GFO, EC 1.1.99.28), and we proposed that these proteins constitute a new protein family (10).…”

“…The cloning of cDNAs for dimeric DDs of human and the above four animals (10) revealed that the enzymes of these species with 82-94% sequence identities show no homology with the monomeric DDs and cis-dihydrodiol dehydrogenases, which belong to the aldo-keto reductase family (11) and short-chain dehydrogenase/reductase family (12), respectively. However, dimeric DD shows sequence similarity to putative gene products of microorganisms and Zymomonas mobilis glucose-fructose oxidoreductase (GFO, EC 1.1.99.28), and we proposed that these proteins constitute a new protein family (10). Crystal structure of NADP ϩ -bound GFO has been solved and suggested that two tyrosines at positions 217 and 296 are candidates for the catalytic residue (13).…”

Cloning, Expression, and Characterization of Bacterial l-Arabinose 1-Dehydrogenase Involved in an Alternative Pathway of l-Arabinose Metabolism

“…Amino Acid Sequence Analysis of L-Arabinose 1-DehydrogenaseProtein-BLAST analysis revealed that L-arabinose 1-dehydrogenase is a putative member of the Gfo/Idh/MocA protein family, which includes GFOR (18 -20), D-xylose 1-dehydrogenase (21), dimeric dihydrodiol dehydrogenase (DD) (38), and myo-inositol 2-dehydrogenase (39) (Fig. 7A).…”