Characterization of Two Novel Aldo-Keto Reductases from Arabidopsis: Expression Patterns, Broad Substrate Specificity, and an Open Active-Site Structure Suggest a Role in Toxicant Metabolism Following Stress
Aldo-keto reductases (AKRs) are widely distributed in nature and play numerous roles in the metabolism of steroids, sugars, and other carbonyls. They have also frequently been implicated in the metabolism of exogenous and endogenous toxicants, including those stimulated by stress. Although the Arabidopsis genome includes at least 21 genes with the AKR signature, very little is known of their functions. In this study, we have screened the Arabidopsis thaliana genomic sequence for genes with significant homology to members of the mammalian AKR1 family and identified four homologues for further study. Following alignment of the predicted protein sequences with representatives from the AKR superfamily, the proteins were ascribed not to the AKR1 family but to the AKR4C subfamily, with the individual designations of AKR4C8, AKR4C9, AKR4C10, and AKR4C11. Expression of two of the genes, AKR4C8 and AKR4C9, has been shown to be coordinately regulated and markedly induced by various forms of stress. The genes have been overexpressed in bacteria, and recombinant proteins have been purified and crystallized. Both enzymes display NADPH-dependent reduction of carbonyl compounds, typical of the superfamily, but will accept a very wide range of substrates, reducing a range of steroids, sugars, and aliphatic and aromatic aldehydes/ketones, although there are distinct differences between the two enzymes. We have obtained high-resolution crystal structures of AKR4C8 (1.4 A) and AKR4C9 (1.25 A) in ternary complexes with NADP(+) and acetate. Three extended loops, present in all AKRs and responsible for defining the cofactor- and substrate-binding sites, are shorter in the 4C subfamily compared to other AKRs. Consequently, the crystal structures reveal open and accommodative substrate-binding sites, which correlates with their broad substrate specificity. It is suggested that the primary role of these enzymes may be to detoxify a range of toxic aldehydes and ketones produced during stress, although the precise nature of the principal natural substrates remains to be determined.
[Show abstract][Hide abstract] ABSTRACT: Complementary DNA clones encoding human aflatoxin B(1) aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine. The AKR1C family members proved not to be as widely expressed as the other reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone. As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further. Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting. Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules. Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions.
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis Co-expression Tool, ACT, ranks the genes across a large microarray dataset according to how closely their expression follows the expression of a query gene. A database stores pre-calculated co-expression results for approximately 21,800 genes based on data from over 300 arrays. These results can be corroborated by calculation of co-expression results for user-defined sub-sets of arrays or experiments from the NASC/GARNet array dataset. Clique Finder (CF) identifies groups of genes which are consistently co-expressed with each other across a user-defined co-expression list. The parameters can be altered easily to adjust cluster size and the output examined for optimal inclusion of genes with known biological roles. Alternatively, a Scatter Plot tool displays the correlation coefficients for all genes against two user-selected queries on a scatter plot which can be useful for visual identification of clusters of genes with similar r-values. User-input groups of genes can be highlighted on the scatter plots. Inclusion of genes with known biology in sets of genes identified using CF and Scatter Plot tools allows inferences to be made about the roles of the other genes in the set and both tools can therefore be used to generate short lists of genes for further characterization. ACT is freely available at www.Arabidopsis.leeds.ac.uk/ACT.
Nucleic Acids Research 08/2006; 34(Web Server issue):W504-9. DOI:10.1093/nar/gkl204 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3alpha-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP(+) (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP(+) controlled k(cat), whereas the chemical event also contributed to this term. k(cat) was insensitive to [(2)H]NADPH, whereas (D)k(cat)/K(m) and the (D)k(lim) (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated k(cat), whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall K(eq) favored reduction. The kinetic Haldane and free energy diagram confirmed that K(eq) was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.
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