The explosive trinitrotoluene (TNT) induces gene expression of carbonyl reductase in the blue mussel (Mytilus spp.): a new promising biomarker for sea dumped war relicts?

Millions of tons of all kind of munitions, including mines, bombs and torpedoes have been dumped after World War II in the marine environment and do now pose a new threat to the seas worldwide. Beside the acute risk of unwanted detonation, there is a chronic risk of contamination, because the metal vessels corrode and the toxic and carcinogenic explosives (trinitrotoluene (TNT) and metabolites) leak into the environment. While the mechanism of toxicity and carcinogenicity of TNT and its derivatives occurs through its capability of inducing oxidative stress in the target biota, we had the idea if TNT can induce the gene expression of carbonyl reductase in blue mussels. Carbonyl reductases are members of the short-chain dehydrogenase/reductase (SDR) superfamily. They metabolize xenobiotics bearing carbonyl functions, but also endogenous signal molecules such as steroid hormones, prostaglandins, biogenic amines, as well as sugar and lipid peroxidation derived reactive carbonyls, the latter providing a defence mechanism against oxidative stress and reactive oxygen species (ROS). Here, we identified and cloned the gene coding for carbonyl reductase from the blue mussel Mytilus spp. by a bioinformatics approach. In both laboratory and field studies, we could show that TNT induces a strong and concentration-dependent induction of gene expression of carbonyl reductase in the blue mussel. Carbonyl reductase may thus serve as a biomarker for TNT exposure on a molecular level which is useful to detect TNT contaminations in the environment and to perform a risk assessment both for the ecosphere and the human seafood consumer.

Carbonyl reductases from Daphnia are regulated by redox cycling compounds.

Oxidative stress is a major source of reactive carbonyl compounds that can damage cellular macromolecules, leading to so-called carbonyl stress. Aside from endogenously formed carbonyls, including highly reactive short-chain aldehydes and diketones, air pollutants derived from diesel exhaust like 9,10-phenanthrenequinone (PQ) can amplify oxidative stress by redox cycling, causing tissue damage. Carbonyl reductases (CRs), which are inducible in response to ROS, represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The microcrustacean Daphnia is an ideal model organism to investigate the function of CRs because of its unique equipment with even four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Cloning and catalytic characterization of two carbonyl reductases CR1 and CR3 from D. magna and D. pulex arenata revealed that both proteins reductively metabolize aromatic dicarbonyls (e.g., menadione, PQ) and aliphatic α-diketones (e.g., 2,3-hexanedione), while sugar-derived aldehydes (methylglyoxal, glyoxal) and lipid peroxidation products such as acrolein and butanal were poor substrates, indicating no physiological function in the metabolism of short-chain aldehydes. Treatment of D. magna with redox cyclers like menadione and the pesticide paraquat led to an upregulation of CR1 and CR3 mRNA, suggesting a role in oxidative stress defense. Further studies are needed to investigate their potential to serve as novel biomarkers for oxidative stress in Daphnia.

Crystal structure and catalytic characterization of the dehydrogenase/reductase SDR family member 4 (DHRS4) from Caenorhabditis elegans.

The human dehydrogenase/reductase SDR family member 4 (DHRS4) is a tetrameric protein that is involved in the metabolism of several aromatic carbonyl compounds, steroids, and bile acids. The only invertebrate DHRS4 that has been characterized to date is that from the model organism Caenorhabditis elegans. We have previously cloned and initially characterized this protein that was recently annotated as DHRS4_CAEEL in the UniProtKB database. Crystallization and X-ray diffraction studies of the full-length DHRS4_CAEEL protein in complex with diacetyl revealed its tetrameric structure and showed that two subunits are connected via an intermolecular disulfide bridge that is formed by N-terminal cysteine residues (Cys5) of each protein chain, which increases the enzymatic activity. A more detailed biochemical and catalytic characterization shows that DHRS4_CAEEL shares some properties with human DHRS4 such as relatively low substrate affinities with aliphatic α-diketones and a preference for aromatic dicarbonyls such as isatin, with a 30-fold lower Km value compared with the human enzyme. Moreover, DHRS4_CAEEL is active with aliphatic aldehydes (e.g. hexanal), while human DHRS4 is not. Dehydrogenase activity with alcohols was only observed with aromatic alcohols. Protein thermal shift assay revealed a stabilizing effect of phosphate buffer that was accompanied by an increase in catalytic activity of more than two-fold. The study of DHRS4 homologs in simple lineages such as C. elegans may contribute to our understanding of the original function of this protein that has been shaped by evolutionary processes in the course of the development from invertebrates to higher mammalian species. DATABASE: Structural data are available in the PDB under the accession numbers 5OJG and 5OJI.

Transcriptional regulation of human and murine short-chain dehydrogenase/reductases (SDRs) - an in silico approach.

Numerous physiological functions of the body are controlled by endogenous (e.g. steroids, retinoids, lipid mediators) or exogenous molecules (e.g. drugs, xenobiotics) that bind to transcription factors (TF). The biosynthesis and catabolism of these signaling molecules depend, apart from CYPs, on enzymes belonging to the short-chain dehydrogenase/reductase (SDR) superfamily. Moreover, the contribution of SDRs to the metabolism of therapeutic drugs and xenobiotics is increasingly recognized. However, only scarce information exists regarding the transcriptional regulation of most SDR proteins. This work aims to illustrate the role of nuclear receptors (NR) and TF related to oxidative stress, inflammation, hypoxia, and xenobiotics in the regulation of selected human and murine SDRs that play crucial roles in steroid, retinoid, eicosanoid, fatty acid, and xenobiotic metabolism. These include, for example, 17ß-hydroxysteroid dehydrogenases, retinol dehydrogenases, and carbonyl reductases. Because existing experimental data are limited, an in silico analysis (TRANSFAC(®) Professional database) of the 5'-upstream sequences for putative response elements was performed. Experimental and in silico data suggest that pharmaceutical, environmental, or dietary NR ligands may alter SDR-mediated retinoid, steroid, and xenobiotic metabolism, likely affecting basic cellular events like energy expenditure, cell proliferation/differentiation, or aging processes. Also, some SDRs are possibly induced by their own substrates. Further experimental work is urgently needed to fully understand the NR-mediated transcriptional regulation of SDRs. This is essential for deducing their possible involvement in drug side effects and will help to identify new substrates and further physiological functions of these SDRs.

Human DCXR - another 'moonlighting protein' involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility?

Dicarbonyl/L-xylulose reductase (DCXR; SDR20C1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily catalyzes the reduction of α-dicarbonyl compounds and monosaccharides. Its role in the metabolism of L-xylulose has been known since 1970, when essential pentosuria was found to be associated with DCXR deficiency. Despite its early discovery, our knowledge about the role of human DCXR in normal physiology and pathophysiology is still incomplete. Sporadic studies have demonstrated aberrant expression in several cancers, but their physiological significance is unknown. In reproductive medicine, where DCXR is commonly referred to as 'sperm surface protein P34H', it serves as marker for epididymal sperm maturation and is essential for gamete interaction and successful fertilization. DCXR exhibits a multifunctional nature, both acting as a carbonyl reductase and also performing non-catalytic functions, possibly resulting from interactions with other proteins. Recent observations associate DCXR with a role in cell adhesion, pointing to a novel function involving tumour progression and possibly metastasis. This review summarizes the current knowledge about human DCXR and its orthologs from mouse and Caenorhabditis elegans (DHS-21) with an emphasis on its multifunctional characteristics. Due to its close structural relationship with DCXR, carbonyl reductase 2 (Cbr2), a tetrameric enzyme found in several non-primate species is also discussed. Similar to human DCXR, Cbr2 from golden hamster (P26h) and cow (P25b) is essential for sperm-zona pellucida interaction and fertilization. Because of the apparent similarity of these two proteins and the inconsistent use of alternative names previously, we provide an overview of the systematic classification of DCXR and Cbr2 and a phylogenetic analysis to illustrate their ancestry.

Genome sequence of Comamonas testosteroni ATCC 11996, a representative strain involved in steroid degradation.

Comamonas testosteroni strains belong to the family of Comamonadaceae and are known for their ability to utilize steroid compounds as carbon source. Here, we present the draft genome sequence of strain ATCC 11996, with a G+C content of 61.48%.

Short-chain dehydrogenases/reductases in cyanobacteria.

The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria.

Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including bacteria and archaea, suggesting that steroid metabolism is an evolutionarily conserved mechanism that might serve different functions such as nutrient supply and signaling. Article from a special issue on steroids and microorganisms.

Bioinformatic and biochemical characterization of DCXR and DHRS2/4 from Caenorhabditis elegans.

Several reductases belonging to the large enzyme superfamily of the short-chain dehydrogenases/reductases (SDR) are involved in the reductive metabolism of carbonyl containing xenobiotics. In order to characterize the human enzymes dicarbonyl/l-xylulose reductase (DCXR), and dehydrogenase/reductase members 2 and 4 (DHRS2, DHRS4) in terms of metabolism of xenobiotics, orthologues from the model organism Caenorhabditis elegans (C. elegans) were identified by using hidden Markov models that were developed in the present study. Accordingly, we describe the characterization of proteins from C. elegans as orthologous to the human enzymes DCXR and DHRS2/4 using a combined approach of bioinformatic and biochemical methods. With the hidden Markov model based system we identified the C. elegans proteins SDR20C18, SDR25C21 and SDR25C22 as being homologous to the human enzymes DCXR, and DHRS2 or DHRS4, respectively. After cloning and overexpression of these three C. elegans genes in Escherichia coli we could purify SDR20C18 and SDR25C22 as soluble proteins by Ni-affinity chromatography, whereas recombinant SDR25C21 was only found in inclusion bodies. Both SDR20C18 (UniProtAcc: Q21929) and SDR25C22 (UniProtAcc: Q93790) were tested with a variety of xenobotic carbonyl compounds as substrates. A comparison of the catalytic activities of SDR20C18 and SDR25C22 with well-known substrates of the human forms revealed that SDR20C18 is the DCXR-orthologue enzyme to the human enzyme and that SDR25C22 might be a DHRS2/4 homologue. Due to their high sequence identity, it was so far not possible to distinguish between SDR25C22 and the human DHRS2/4 proteins by means of sequence analysis alone. However, the study of homologue genes in the model organism C. elegans can provide valuable information on the putative physiological role of the corresponding human form.

Proteasome inhibitors MG-132 and bortezomib induce AKR1C1, AKR1C3, AKR1B1, and AKR1B10 in human colon cancer cell lines SW-480 and HT-29.

Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1-1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1-1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the phenomenon of multidrug resistance upon cancer chemotherapy.

Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3.

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.

The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase.

Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 µM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE.

Regulation of human carbonyl reductase 3 (CBR3; SDR21C2) expression by Nrf2 in cultured cancer cells.

Carbonyl reduction is a central metabolic process that controls the level of key regulatory molecules as well as xenobiotics. Carbonyl reductase 3 (CBR3; SDR21C2), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, has been poorly characterized so far, and the regulation of its expression is a complete mystery. Here, we show that CBR3 expression is regulated via Nrf2, a key regulator in response to oxidative stress. In human cancer cell lines, CBR3 mRNA was expressed differentially, ranging from very high (A549, lung) to very low (HT-29, colon; HepG2, liver) levels. CBR3 protein was highly expressed in SW-480 (colon) cells but was absent in HCT116 (colon) and HepG2 cells. CBR3 mRNA could be induced in HT-29 cells by Nrf2 agonists [sulforaphane (SUL, 7-fold) and diethyl maleate (DEM, 4-fold)] or hormone receptor ligand Z-guggulsterone (5-fold). Aryl hydrocarbon receptor agonist B[k]F failed to induce CBR3 mRNA after incubation for 8 h but elevated CBR3 levels after 24 h, most likely mediated by B[k]F metabolites that can activate Nrf2 signaling. Inhibition of Nrf2-activating upstream kinase MEK/ERK by PD98059 weakened DEM-mediated induction of CBR3 mRNA. Proteasome inhibitors MG-132 (5 µM) and bortezomib (50 nM) dramatically increased the level of CBR3 mRNA, obviously because of the increase in the level of Nrf2 protein. While siRNA-mediated knockdown of Nrf2 led to a decrease in the level of CBR3 mRNA in A549 cells (30% of control), Keap1 knockdown increased the level of CBR3 mRNA expression in HepG2 (9.3-fold) and HT-29 (2.7-fold) cells. Here, we provide for the first time evidence that human CBR3 is a new member of the Nrf2 gene battery.

Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by site-directed mutagenesis.

Human carbonyl reductase is a member of the short-chain dehydrogenase/reductase (SDR) protein superfamily and is known to play an important role in the detoxification of xenobiotics bearing a carbonyl group. The two monomeric NADPH-dependent human isoforms of cytosolic carbonyl reductase CBR1 and CBR3 show a sequence similarity of 85% on the amino acid level, which is definitely high if compared to the low similarities usually observed among other members of the SDR superfamily (15-30%). Despite the sequence similarity and the similar features found in the available crystal structures of the two enzymes, CBR3 shows only low or no activity towards substrates that are metabolised by CBR1. This surprising substrate specificity is still not fully understood. In the present study, we introduced several point mutations and changed sequences of up to 17 amino acids of CBR3 to the corresponding amino acids of CBR1, to gather insight into the catalytic mechanism of both enzymes. Proteins were expressed in Escherichia coli and purified by Ni-affinity chromatography. Their catalytic properties were then compared using isatin and 9,10-phenanthrenequinone as model substrates. Towards isatin, wild-type CBR3 showed a catalytic efficiency of 0.018 microM(-1)min(-1), whereas wild-type CBR1 showed a catalytic efficiency of 13.5 microM(-1)min(-1). In particular, when nine residues (236-244) in the vicinity of the catalytic center and a proline (P230) in CBR3 were mutated to the corresponding residues of CBR1 a much higher k(cat)/K(m) value (5.7 microM(-1)min(-1)) towards isatin was observed. To gain further insight into the protein-ligand binding process, docking simulations were perfomed on this mutant and on both wild-type enzymes (CBR1 and CBR3). The theoretical model of the mutant was ad hoc built by means of standard comparative modelling.

The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative.

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with presently over 46,000 members. In phylogenetic comparisons, members of this superfamily show early divergence where the majority have only low pairwise sequence identity, although sharing common structural properties. The SDR enzymes are present in virtually all genomes investigated, and in humans over 70 SDR genes have been identified. In humans, these enzymes are involved in the metabolism of a large variety of compounds, including steroid hormones, prostaglandins, retinoids, lipids and xenobiotics. It is now clear that SDRs represent one of the oldest protein families and contribute to essential functions and interactions of all forms of life. As this field continues to grow rapidly, a systematic nomenclature is essential for future annotation and reference purposes. A functional subdivision of the SDR superfamily into at least 200 SDR families based upon hidden Markov models forms a suitable foundation for such a nomenclature system, which we present in this paper using human SDRs as examples.