Characterization of the human aldehyde reductase gene and promoter.

Aldehyde reductase (EC 1.1.1.2; AKR1A1) is involved in the reduction of biogenic and xenobiotic aldehydes and is present in virtually every tissue. To study the regulation of its expression, the human aldehyde reductase gene and promoter were cloned and characterized. The protein coding region consists of eight exons, with two additional upstream exons, separated by a large intron of 9.4 kb, that code for the 5' untranslated region of the mRNA. Two mRNA transcripts that encode the same protein and that originate from alternative splicing were identified. The shorter transcript is the major form as shown by Northern blots and reverse transcription-PCR experiments. Northern blots of multiple tissues indicate that aldehyde reductase mRNA is present in all tissues examined and is most abundant in kidney, liver, and thyroid, which is consistent with the tissue enzyme distribution. The two mRNA transcripts do not exhibit differential tissue distribution. A construct containing a promoter region insert in a pGL3 vector drives transcription of a luciferase reporter gene and is 290-fold more active than a control vector without insert in transfected HepG2 cells. The activity of the full promoter construct is comparable to that of a pGL3 vector containing the SV40 promoter with an enhancer. The promoter does not contain a TATA box, but contains multiple GC-rich islands and exhibits bidirectional activity in transfection studies. The major active promoter element was localized by nested deletions and mutations to a DNA element (TGCAAT, -59 to -54) that presumptively binds the transcription factor CHOP [CAAT enhancer binding protein (C/EBP) homologous protein]. Comparison of the aldehyde reductase gene structure to all other characterized human genes of the aldo-keto reductase superfamily (aldose reductase, bile acid binder, and type I and type II 3alpha-hydroxysteroid dehydrogenases) indicates that it is more distantly related to these genes than they are among themselves.

The C-terminal loop of aldehyde reductase determines the substrate and inhibitor specificity.

Human aldehyde reductase has a preference for carboxyl group-containing negatively charged substrates. It belongs to the NADPH-dependent aldo-keto reductase superfamily whose members are in part distinguished by unique C-terminal loops. To probe the role of the C-terminal loops in determining substrate specificities in these enzymes, two arginine residues, Arg308 and Arg311, located in the C-terminal loop of aldehyde reductase, and not found in any other C-terminal loop, were replaced with alanine residues. The catalytic efficiency of the R311A mutant for aldehydes containing a carboxyl group is reduced 150-250-fold in comparison to that of the wild-type enzyme, while substrates not containing a negative charge are unaffected. The R311A mutant is also significantly less sensitive to inhibition by dicarboxylic acids, indicating that Arg311 interacts with one of the carboxyl groups. The inhibition pattern indicates that the other carboxyl group binds to the anion binding site formed by Tyr49, His112, and the nicotinamide moiety of NADP+. The correlation between inhibitor potency and the length of the dicarboxylic acid molecules suggests a distance of approximately 10 A between the amino group of Arg311 and the anion binding site in the aldehyde reductase molecule. The sensitivity of inhibition of the R311A mutant by several commercially available aldose reductase inhibitors (ARIs) was variable, with tolrestat and zopolrestat becoming more potent inhibitors (30- and 5-fold, respectively), while others remained the same or became less potent. The catalytic properties, substrate specificity, and susceptibility to inhibition of the R308A mutant remained similar to that of the wild-type enzyme. The data provide direct evidence for C-terminal loop participation in determining substrate and inhibitor specificity of aldo-keto reductases and specifically identifies Arg311 as the basis for the carboxyl-containing substrate preference of aldehyde reductase.

Mechanism of human aldehyde reductase: characterization of the active site pocket.

Human aldehyde reductase is a NADPH-dependent aldo-keto reductase that is closely related (65% identity) to aldose reductase, an enzyme involved in the pathogenesis of some diabetic and galactosemic complications. In aldose reductase, the active site residue Tyr48 is the proton donor in a hydrogen-bonding network involving residues Asp43/Lys77, while His110 directs the orientation of substrates in the active site pocket. Mutation of the homologous Tyr49 to phenylalamine or histidine (Y49F or Y49H) and of Lys79 to methionine (K79M) in aldehyde reductase yields inactive enzymes, indicating similar roles for these residues in the catalytic mechanism of aldehyde reductase. A H112Q mutant aldehyde reductase exhibited a substantial decrease in catalytic efficiency (kcat/Km) for hydrophilic (average 150-fold) and aromatic substrates (average 4200-fold) and 50-fold higher IC50 values for a variety of inhibitors than that of the wild-type enzyme. The data suggest that His112 plays a major role in determining the substrate specificity of aldehyde reductase, similar to that shown earlier for the homologous His110 in aldose reductase [Bohren, K. M., et. al. (1994) Biochemistry 33, 2021-2032]. Mutation of Ile298 or Val299 affected the kinetic parameters to a much lesser degree. Unlike native aldose reductase, which contains a thiol-sensitive Cys298, neither the I298C or V299C mutant exhibited any thiol sensitivity, suggesting a geometry of the active site pocket different from that in aldose reductase. Also different from aldose reductase, the detection of a significant primary deuterium isotope effect on kcat (1.48 +/- 0.02) shows that nucleotide exchange is only partially rate-limiting. Primary substrate and solvent deuterium isotope effects on the H112Q mutant suggest that hydride and proton transfers occur in two discrete steps with hydride transfer taking place first. Dissociation constants and spectroscopic and fluorimetric properties of nucleotide complexes with various mutants suggest that, in addition to Tyr49 and His112, Lys79 plays a hitherto unappreciated role in nucleotide binding. The mode of inhibition of aldehyde reductase by aldose reductase inhibitors (ARIs) is generally similar to that of aldose reductase and involves binding to the E:NADP+ complex, as shown by kinetic and direct inhibitor-binding experiments. The order of ARI potency was AL1576 (Ki = 60 nM) > tolrestat > ponalrestat > sorbinil > FK366 > zopolrestat > alrestatin (Ki = 148 microM). Our data on aldehyde reductase suggest that the active site pocket significantly differs from that of aldose reductase, possibly due to the participation of the C-terminal loop in its formation.

Kinetic mechanism of ketoreductase activity of prostaglandin F synthase from bovine lung.

The kinetic mechanism of ketoreductase activity of bovine lung prostaglandin F synthase, expressed in E. coli, was investigated. Data on initial velocity and radioisotope exchange between [3H]prostaglandin D2 and 9 alpha,11 beta-prostaglandin F2 suggest that the enzyme obeys the ping-pong mechanism. Using a fluorescence technique we obtained a binding constant of 3 microM for NADPH. This is in close correlation with the kinetically determined intrinsic Michaelis constant for NADPH. Activation energy of the redox process was determined from the temperature dependence of maximal velocities for nitrobenzaldehyde and menadione and was found to be 119 and 96 kJ/mol, respectively.

A lung type prostaglandin F synthase is expressed in bovine liver: cDNA sequence and expression in E. coli.

Using the cDNA of bovine lung prostaglandin F synthase (EC 1.1.1.2) as a probe, we isolated a clone from a bovine liver cDNA library which differed in only eleven nucleotides from the probe. The corresponding protein contained three amino acid substitutions, including a leucine residue which is conserved throughout all aldo-keto reductases. We inserted the liver cDNA into expression vector pUC19 and expressed the recombinant liver enzyme in E.coli. The purified liver enzyme reduced prostaglandin H2 as well as prostaglandin D2 and various carbonyl compounds. The high relative activity against prostaglandin H2 in combination with a high Km value for prostaglandin D2 identified this liver enzyme as a lung type prostaglandin F synthase. However, the binding constant for NADPH of the liver enzyme was 3.5 fold higher than that of lung prostaglandin F synthase.