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.

Structural and functional features of dimeric dihydrodiol dehydrogenase

“…The purified enzymes are composed of identical subunits with molecular weights of 32 -36 kDa. The cDNAs for the enzymes of the above animal species, Cynomolgus monkey and humans were cloned, and their deduced amino acid sequences reveal that their subunits consist of 334 or 335 amino acids [33]. These studies indicate a species-specific tissue distribution of DHDH.…”

“…Amongst SDR enzymes this can include the coenzyme-binding motif of GlyxxxGlyxGly (where x is any variable amino acid residue), creating a bab Rossmann fold [53], or the active-site sequence motif Tyr-X-X-XLys [54,55]. DHDHs have a high degree of interspecies sequence similarity (82 -94 %), low sequence similarity (17 -41 %) with 20 putative gene products of micro-organisms, including glucose-fructose oxido-reductase (GFO, EC 1.1.99.28) from Zymomonas mobilis, and no significant homology (or structural motif identity) with members of the SDR or AKR enzymes [33,56]. While the specific sequence motifs indicating the presence of a Rossmann fold in SDRs are not found in DHDH, the tertiary structure clearly indicates its presence, suggesting that this nascent group of enzymes may be best assessed structurally to denote new or potential family members and therefore identify new consensus sequences that would best characterize these proteins.…”

“…These studies indicate a species-specific tissue distribution of DHDH. The enzyme is ubiquitously distributed in pigs [23] and dogs [37], whereas it is specifically expressed in kidney of monkeys [38], lens and intestine of rabbits, and intestine of humans [33]. DHDH has not been detected in mouse liver and kidney [15,18,39].…”

“…8). The Rossmann fold amongst DHDHs is characterized with the fingerprint sequence HX 2 HX 16 EKP (residues 76 -98) [33], while a consensus sequence involving the bacterial oxido-reductases of GFO, AFR and TMO forms a smaller motif of GX 2 VXCEKP (residues 90 -98 in DD) resulting from the absence of His76 in AFR, GFO and TMO and His79 in the TMO sequences. Earlier mutagenesis studies based on knowledge of the structure of GFO and the sequence of DHDH from Japanese monkey kidney have identified several conserved catalytic and dinucleotide binding residues [46], including His79, which functions as a coenzyme binding residue, and Tyr180, which is critical for the reaction mechanism.…”

“…There have been several excellent reviews on the structures and functions of the enzymes in the AKR superfamily [29 -32]. On the other hand, the physiological substrates and amino acid sequences of dimeric DDs have recently been identified [33,34] and the crystal structure of the monkey enzyme recently determined [35]. These studies reveal that dimeric DD is dissimilar to the enzymes of the SDR and AKR superfamilies, but is reminiscent of oxido-reductases found in bacterial species and together form a new and unique family of oxido-reductases.…”