Acceleration of an aldo-keto reductase by minimal loop engineering.
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ABSTRACT:
SUBMITTER: Krump C
PROVIDER: S-EPMC4064709 | biostudies-literature | 2014 Jul
REPOSITORIES: biostudies-literature
Publications
Acceleration of an aldo-keto reductase by minimal loop engineering.
Krump C C Vogl M M Brecker L L Nidetzky B B Kratzer R R
Protein engineering, design & selection : PEDS 20140701 7
Aldo-keto reductases tighten coenzyme binding by forming a hydrogen bond across the pyrophosphate group of NAD(P)(H). Mutation of the hydrogen bonding anchor Lys24 in Candida tenuis xylose reductase prevents fastening of the "safety belt" around NAD(H). The loosened NAD(H) binding leads to increased turnover numbers (k(cat)) for reductions of bulky-bulky ketones at constant substrate and coenzyme affinities (i.e. K(m Ketone), K(m NADH)). ...[more]
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Project description:Aldo-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17?-hydroxysteroid dehydrogenase activity) as well as 3-keto-acyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues.
| S-EPMC4358287 | biostudies-literature
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.
| S-EPMC3206293 | biostudies-literature
Project description:AKR1C3 is a drug target in hormonal and hormonal independent malignancies and acts as a major peripheral 17?-hydroxysteroid dehydrogenase to yield the potent androgens testosterone and dihydrotestosterone, and as a prostaglandin (PG) F synthase to produce proliferative ligands for the PG FP receptor. AKR1C3 inhibitors may have distinct advantages over existing therapeutics for the treatment of castration resistant prostate cancer, breast cancer and acute myeloid leukemia. Area covered: This article reviews the patent literature on AKR1C3 inhibitors using SciFinder which identified inhibitors in the following chemical classes: N-phenylsulfonyl-indoles, N-(benzimidazoylylcarbonyl)- N-(indoylylcarbonyl)- and N-(pyridinepyrrolyl)- piperidines, N-benzimidazoles and N-benzindoles, repurposed nonsteroidal antiinflammatory drugs (indole acetic acids, N-phenylanthranilates and aryl propionic acids), isoquinolines, and nitrogen and sulfur substituted estrenes. The article evaluates inhibitor AKR potency, specificity, efficacy in cell-based and xenograft models and clinical utility. The advantage of bifunctional compounds that either competitively inhibit AKR1C3 and block its androgen receptor (AR) coactivator function or act as AKR1C3 inhibitors and direct acting AR antagonists are discussed. Expert opinion: A large number of potent and selective inhibitors of AKR1C3 have been described however, preclinical optimization, is required before their benefit in human disease can be assessed.
| S-EPMC5724044 | biostudies-literature
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.
| S-EPMC1218714 | biostudies-other
Project description:Regulation of glucose homeostasis is a fundamental process to maintain blood glucose at a physiological level, and its dysregulation is associated with the development of several metabolic diseases. Here, we report on a zebrafish mutant for Aldo-keto-reductase 1a1b (akr1a1b) as a regulator of gluconeogenesis. Adult akr1a1b -/- mutant zebrafish developed fasting hypoglycemia, which was caused by inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression as rate-limiting enzyme of gluconeogenesis. Subsequently, glucogenic amino acid glutamate as substrate for gluconeogenesis accumulated in the kidneys, but not in livers, and induced structural and functional pronephros alterations in 48-hpf akr1a1b -/- embryos. Akr1a1b -/- mutants displayed increased nitrosative stress as indicated by increased nitrotyrosine, and increased protein-S-nitrosylation. Inhibition of nitrosative stress using the NO synthase inhibitor L-NAME prevented kidney damage and normalized PEPCK expression in akr1a1b -/- mutants. Thus, the data have identified Akr1a1b as a regulator of gluconeogenesis in zebrafish and thereby controlling glucose homeostasis.
| S-EPMC7683270 | biostudies-literature
Project description:The abstract of the associated publication (Selga E, Noé V, Ciudad CJ. Biochemical Pharmacology, 2008) is the following: While studying differentially expressed genes between sensitive and 10-5 M Methotrexate (MTX) resistant HT29 human colon cancer cells, we identified some members of the aldo-keto reductase (AKR) superfamily. The study was followed with the member AKR1C1 (EC 1.1.1.213), validating its increase in mRNA and protein levels in MTX resistant cells. The genomic content for AKR1C1 remained unchanged between sensitive and resistant cells, thereby excluding a mechanism of AKR1C1 gene amplification. Thus, we cloned the AKR1C1 human promoter and performed luciferase experiments that revealed a transcriptional regulation of the gene in the resistant cells. Computational studies showed a putative binding site for the transcription factor Sp1. The co-transfection of Sp1 or Sp3 with different constructs of AKR1C1 promoter deletions, including and excluding the proximal GC-box, demonstrated a key role for these factors in regulating AKR1C1 transcriptional activity. Gel-shift assays revealed an increase in Sp1 and Sp3 binding in resistant compared to sensitive cells, without differences in Sp1 protein levels. Dephosphorylation of the extracts coincided with a decrease in Sp1 binding, which is consistent with a process of regulation of Sp1 by phosphorylation. We also investigated the possible relationship between AKR1C1 expression and MTX action. Overexpression of AKR1C1 counteracted the S-phase accumulation of cells and apoptosis caused by MTX treatment. This suggests a role of AKR1C1 in cell proliferation. Finally, overexpression of AKR1C1 in MTX sensitive HT29 cells conferred resistance to the chemotherapeutic agent and silencing of AKR1C1 by means of iRNA technology sensitized the cells to MTX. Keywords: DHFR, Methotrexate, drug-resistance
2007-12-30 | GSE9412 | GEO
Project description:Aldo-keto reductases (AKRs) are monomeric NAD(P)(H)-dependent oxidoreductases that play pivotal roles in the biosynthesis and metabolism of steroids in humans. AKR1C enzymes acting as 3-ketosteroid, 17-ketosteroid, and 20-ketosteroid reductases are involved in the prereceptor regulation of ligands for the androgen, estrogen, and progesterone receptors and are considered drug targets to treat steroid hormone-dependent malignancies and endocrine disorders. In contrast, AKR1D1 is the only known steroid 5?-reductase and is essential for bile-acid biosynthesis, the generation of ligands for the farnesoid X receptor, and the 5?-dihydrosteroids that have their own biological activity. In this review we discuss the crystal structures of these AKRs, their kinetic and catalytic mechanisms, AKR genomics (gene expression, splice variants, polymorphic variants, and inherited genetic deficiencies), distribution in steroid target tissues, roles in steroid hormone action and disease, and inhibitor design.
| S-EPMC6405412 | biostudies-literature
Project description:In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta.
| S-EPMC5466437 | biostudies-literature
Project description:BACKGROUND:In nature, termites can be considered as a model biological system for biofuel research based on their remarkable efficiency for lignocellulosic biomass conversion. Redox enzymes are of interest in second-generation ethanol production because they promote synergic enzymatic activity with classical hydrolases for lignocellulose saccharification and inactivate fermentation inhibitory compounds produced after lignocellulose pretreatment steps. RESULTS:In the present study, the biochemical and structural characteristics of the Coptotermes gestroi aldo-keto reductase (CgAKR-1) were comprehensively investigated. CgAKR-1 displayed major structural differences compared with others AKRs, including the differences in the amino acid composition of the substrate-binding site, providing basis for classification as a founding member of a new AKR subfamily (family AKR1 I). Immunolocalization assays with anti-CgAKR-1 antibodies resulted in strong fluorescence in the salivary gland, proventriculus, and foregut. CgAKR-1 supplementation caused a 32% reduction in phenolic aldehydes, such as furfural, which act as fermentation inhibitors of hemicellulosic hydrolysates, and improved ethanol fermentation by the xylose-fermenting yeast Scheffersomyces stipitis by 45%. We observed synergistic enzymatic interactions between CgAKR-1 and commercial cellulosic cocktail for sugarcane bagasse saccharification, with a maximum synergism degree of 2.17 for sugar release. Our data indicated that additive enzymatic activity could be mediated by reactive oxygen species because CgAKR-1 could produce hydrogen peroxide. CONCLUSION:In summary, we identified the founding member of an AKRI subfamily with a potential role in the termite digestome. CgAKR-1 was found to be a multipurpose enzyme with potential biotechnological applications. The present work provided a basis for the development and application of integrative and multipurpose enzymes in the bioethanol production chain.
| S-EPMC5209882 | biostudies-literature
Project description:Human aldo-keto reductase 1B10 (AKR1B10) is overexpressed in many cancer types and is involved in chemoresistance. This makes AKR1B10 to be an interesting drug target and thus many enzyme inhibitors have been investigated. High-resolution crystallographic structures of AKR1B10 with various reversible inhibitors were deeply analyzed and compared to those of analogous complexes with aldose reductase (AR). In both enzymes, the active site included an anion-binding pocket and, in some cases, inhibitor binding caused the opening of a transient specificity pocket. Different structural conformers were revealed upon inhibitor binding, emphasizing the importance of the highly variable loops, which participate in the transient opening of additional binding subpockets. Two key differences between AKR1B10 and AR were observed regarding the role of external loops in inhibitor binding. The first corresponded to the alternative conformation of Trp112 (Trp111 in AR). The second difference dealt with loop A mobility, which defined a larger and more loosely packed subpocket in AKR1B10. From this analysis, the general features that a selective AKR1B10 inhibitor should comply with are the following: an anchoring moiety to the anion-binding pocket, keeping Trp112 in its native conformation (AKR1B10-like), and not opening the specificity pocket in AR.
| S-EPMC8708191 | biostudies-literature