Structural and Enzymatic Properties of a Gastric NADP(H)- dependent and Retinal-active Alcohol Dehydrogenase

Abstract

A class IV-type, gastric alcohol dehydrogenase (ADH) has been purified from frog (Rana perezi) tissues, meaning detection of this enzyme type also in nonmammalian vertebrates. However, the protein is unique among vertebrate ADHs thus far characterized in having preference for NADP ؉ rather than NAD ؉ . Similarly, it deviates structurally from other class IV ADHs and has a phylogenetic tree position outside that of the conventional class IV cluster. The NADP ؉ preference is structurally correlated with a replacement of Asp-223 of all other vertebrate ADHs with Gly-223, largely directing the coenzyme specificity. This residue replacement is expected metabolically to correlate with a change of the reaction direction catalyzed, from preferential alcohol oxidation to preferential aldehyde reduction. This is of importance in cellular growth regulation through retinoic acid formed from retinol/retinal precursors because the enzyme is highly efficient in retinal reduction (k cat /K m ‫؍‬ 3.4⅐10 4 mM ؊1 min ؊1 ). Remaining enzymatic details are also particular but resemble those of the human class I/class IV enzymes. However, overall structural relationships are distant (58 -60% residue identity), and residues at substrate binding and coenzyme binding positions are fairly deviant, reflecting the formation of the new activity. The results are concluded to represent early events in the duplicatory origin of the class IV line or of a separate, class IV-type line. In both cases, the novel enzyme illustrates enzymogenesis of classes in the ADH system. The early origin (with tetrapods), the activity (with retinoids), and the specific location of this enzyme (gastric, like the gastric and epithelial location of the human class IV enzyme) suggest important functions of the class IV ADH type in vertebrates.

A Vertebrate Aldo-keto Reductase Active with Retinoids and Ethanol

“…In this study we have recognized an activity in the upper digestive tract of chicken, similar to that of ADH from the MDR family previously identified in mammals (11,35) and amphibians (17). However, this avian enzyme was not an ADH of the MDR family, but, surprisingly, an AKR.…”

“…Protein Sequence Analysis-Protein for sequence analysis was reduced with dithiothreitol and carboxymethylated with iodoacetate as described for ADH (17). Aliquots were then submitted to proteolytic digestions with trypsin, Lys-C protease, N-Asp protease, or Glu-C protease in 0.1 M ammonium bicarbonate or to treatment with CNBr in 70% formic acid.…”

“…ADH7 has been described only in chicken embryo, exhibiting activity with retinoids (16). ADH8 is an NADP(H)-dependent form with tissue distribution and substrate specificity similar to those of mammalian ADH4, which has been described in amphibians (17). The SDR superfamily contains an alcohol dehydrogenase that accounts for ethanol metabolism in Drosophila (18) and several mammalian forms that exhibit activity with retinoids (15,19,20).…”

Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol dehydrogenases fromSaccharomyces cerevisiaeandRana perezi

“…In our search for an ADH4-type enzyme in amphibians, we found an enzyme with similar kinetic properties to those of ADH4 but, surprisingly, with speci®city towards NADP(H) instead of NAD(H), the common coenzyme for all previously described vertebrate ADHs. This enzyme (ADH8) was isolated from the stomach of R. perezi and exhibited high activity towards retinal (Peralba et al, 1999). In consequence, we proposed a physiological function in retinal reduction for ADH8, which is also supported by the high cellular NADPH: NADP + ratio.…”

“…perezi (ADH8). Wild-type ADH8 cDNA was obtained from isolated poly(A) + RNA and cloned into pBluescript II SK (+) (Peralba et al, 1999). Cloning into pGEX 4T-2 (Amersham Pharmacia Biotech) was performed by ampli®cation of the coding region using the polymerase chain reaction (PCR) with two speci®c primers, RanaL (5 H -TTATAGGATCCATGTGCACTGCGGG-AAAAGAT-3 H ) and RanaR (5 H -CCAC-CTGAATTCTTAGTATATCATAATGCT-TCG-3 H ), including restriction sites (bold) for BamHI and EcoRI, respectively.…”