Dataset Information

Purification from rat liver of a novel constitutively expressed member of the aldo-keto reductase 7 family that is widely distributed in extrahepatic tissues.

ABSTRACT: Antiserum raised against human aflatoxin B(1) aldehyde reductase 1 (hAFAR1) has been used to identify a previously unrecognized rat aldo-keto reductase (AKR). This novel enzyme is designated rat aflatoxin B(1) aldehyde reductase 2 (rAFAR2) and it characteristically migrates faster during SDS/PAGE than does the archetypal ethoxyquin-inducible rAFAR protein (now called rAFAR1). Significantly, rAFAR2 is essentially unreactive with polyclonal antibodies raised against rAFAR1. Besides its distinct electrophoretic and immunochemical properties, rAFAR2 appears to be regulated differently from rAFAR1 as it is expressed in most rat tissues and does not appear to be induced by ethoxyquin. Multiple forms of rAFAR2 have been identified. Anion-exchange chromatography on Q-Sepharose, followed by adsorption chromatography on columns of Matrex Orange A and Cibacron Blue, have been employed to purify rAFAR2 from rat liver cytosol. The Q-Sepharose chromatography step resulted in the resolution of rAFAR2 into three peaks of AKR activity, two of which were purified and shown to be capable of catalysing the reduction of 2-carboxybenzaldehyde, succinic semialdehyde, 4-nitrobenzaldehyde and 9,10-phenathrenequinone. The two most highly purified rAFAR2-containing preparations eluted from the Cibacron Blue column were 91 and 98% homogeneous. Analysis of these by SDS/PAGE indicated that the least anionic (peak CBA5) comprised a polypeptide of 37.0 kDa, whereas the most anionic (peak CBA6) contained two closely migrating polypeptides of 36.8 and 37.0 kDa; by contrast, in the present study, rAFAR1 was estimated by SDS/PAGE to be composed of 38.0 kDa subunits. Final purification of the 37 kDa polypeptide in CBA5 and CBA6 was accomplished by reversed-phase HPLC. Partial proteolysis of the two preparations of the 37 kDa polypeptide with Staphylococcus aureus V8 protease yielded fragments of identical size, suggesting that they represent the product of a single gene. Furthermore, the peptide maps from CBA5 and CBA6 differed substantially from that yielded by rAFAR1, indicating that they are genetically distinct from the inducible reductase. A peptide generated by CNBr digestion of the 37 kDa polypeptide from CBA6 was shown by Edman degradation to share 88% sequence identity with residues Tyr(168)-Leu(183) of rAFAR1. This provides evidence that the rat protein identified by its cross-reactivity with anti-hAFAR1 serum is an additional member of the AKR7 family.

SUBMITTER: Kelly VP

PROVIDER: S-EPMC1221078 | biostudies-other | 2000 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Aldo-keto reductases (AKRs) are phase I metabolising enzymes that catalyse the reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-dependent reduction of carbonyl groups to yield primary and secondary alcohols on a wide range of substrates, including aliphatic and aromatic aldehydes and ketones, ketoprostaglandins, ketosteroids and xenobiotics. In so doing they functionalise the carbonyl group for conjugation (phase II enzyme reactions). Although functionally diverse, AKRs form a protein superfamily based on their high sequence identity and common protein fold, the (alpha/beta) 8 -barrel structure. Well over 150 AKR enzymes, from diverse organisms, have been annotated so far and given systematic names according to a nomenclature that is based on multiple protein sequence alignment and degree of identity. Annotation of non-vertebrate AKRs at the National Center for Biotechnology Information or Vertebrate Genome Annotation (vega) database does not often include the systematic nomenclature name, so the most comprehensive overview of all annotated AKRs is found on the AKR website (http://www.med.upenn.edu/akr/). This site also hosts links to more detailed and specialised information (eg on crystal structures, gene expression and single nucleotide polymorphisms [SNPs]). The protein-based AKR nomenclature allows unambiguous identification of a given enzyme but does not reflect the wealth of genomic and transcriptomic variation that exists in the various databases. In this context, identification of putative new AKRs and their distinction from pseudogenes are challenging. This review provides a short summary of the characteristic features of AKR biochemistry and structure that have been reviewed in great detail elsewhere, and focuses mainly on nomenclature and database entries of human AKRs that so far have not been subject to systematic annotation. Recent developments in the annotation of SNP and transcript variance in AKRs are also summarised.

Project description:The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.

Dataset Information

Purification from rat liver of a novel constitutively expressed member of the aldo-keto reductase 7 family that is widely distributed in extrahepatic tissues.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets