Carbonyl Reductase Research Articles

Enantioselective synthesis of enantiopure chiral alcohols using carbonyl reductases screened from Yarrowia lipolytica.

We aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis. Yarrowia lipolytica carbonyl reductase genes were obtained via homologous sequence amplification strategy. Two carbonyl reductases, YaCRI and YaCRII, were identified and characterized, and used to catalyse the conversion of 2-hydroxyacetophenone (2-HAP) to optically pure (S)-1-phenyl-1,2-ethanediol. Enzymatic assays revealed that YaCRI and YaCRII exhibited specific activities of 6·96Umg-1 (99·8% e.e.) and 7·85Umg-1 (99·9% e.e.), respectively, and showed moderate heat resistance at 40-50°C and acid tolerance at pH 5·0-6·0. An efficient whole-cell two-phase system was established using reductase-expressing recombinant Escherichia coli. The conversion of 2-HAP (20·0gl-1 ) conversion with the solvent of dibutyl phthalate was approximately 70-fold higher than in water. Furthermore, the two recombinant E.coli displayed biocatalyst activity and enantioselectivity towards several different carbonyl compounds, and E.coli BL21 (DE3)/pET-28a-yacrII showed a broad substrate spectrum. A new whole-cell recombinant E.coli-based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed. We proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.

Development of an Efficient and Cost-Effective Enzymatic Process for Production of (R)-[3,5-bis(trifluoromethyl)phenyl] Ethanol Using Carbonyl Reductase Derived from Leifsonia sp. S749.

(R)-[3,5-bis(trifluoromethyl) phenyl] ethanol [(R)-3,5-BTPE] is a crucial chiral intermediate for the synthesis of the NK-1 receptor antagonists aprepitant, rolapitant and fosaprepitant. The carbonyl reductase KR01 from Leifsonia sp. S749, discovered by protein sequence alignment, could convert 3',5'-bis(trifluoromethyl) acetophenone (3,5-BTAP) into (R)-3,5-BTPE with excellent activity and enantioselectivity. In order to enhance the conversion efficiency at high substrate concentrations, the reaction conditions were optimized by response surface analysis. The results showed that 600g/L 3,5-BTAP was bioreduced to (R)-3,5-BTPE (> 99.9% enantiomeric excess) by the recombinant Escherichia coli/pET-28a (+)-KR01 whole cells, with a 98.3% conversion and 59g/L/h productivity under the optimized reaction conditions. In addition, the recombinant E. coli cells could be repeatedly used up to seven times in the reaction mixture containing 90% isopropanol (IPA). This is the highest substrate loading and productivity for the bioreduction of 3,5-BTAP by carbonyl reductase ever reported, and this method represents an efficient and cost-effective process for production of (R)-3,5-BTPE.

Discovery of 5-(2-chloro-4′-(1H-imidazol-1-yl)-[1,1′-biphenyl]-4-yl)-1H-tetrazole as potent and orally efficacious S-nitrosoglutathione reductase (GSNOR) inhibitors for the potential treatment of COPD

Endogenous nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO) serve as reservoir for bioavailable nitric oxide (NO) and mediate NO-based signaling, inflammatory status and smooth muscle function in the lung. GSNOR inhibition increases pulmonary GSNO and induces bronchodilation while reducing inflammation in lung diseases. In this letter, design, synthesis and structure–activity relationships (SAR) of novel imidazole-biaryl-tetrazole based GSNOR inhibitors are described. Many potent inhibitors (30, 39, 41, 42, 44, 45 and 58) were identified with low nanomolar activity (IC50s: <15 nM) along with adequate metabolic stability. Lead compounds 30 and 58 exhibited good exposure and oral bioavailability in mouse pharmacokinetic (PK) study. Compound 30 was selected for further profiling and revealed comparable mouse and rat GSNOR potency, high selectivity against alcohol dehydrogenase (ADH) and carbonyl reductase (CBR1) family of enzymes, low efflux ratio and permeability in PAMPA, a high permeability in CALU-3 assay, significantly low hERG activity and minimal off-target activity. Further, an in vivo efficacy of compound 30 is disclosed in cigarette smoke (CS) induced mouse model for COPD.

Proteomic footprint of ultrasound intensification on sliced dry-cured ham subjected to mild thermal conditions

Ultrasound can intensify the heating process used to correct texture defects in dry-cured hams. The effect of ultrasound-assisted heating on the proteome of sliced dry-cured ham was evaluated. Dry-cured hams with high proteolysis index (PI > 36) were sliced, vacuum packed and subjected to conventional (CV) and ultrasound-assisted (US) thermal treatments. Comparative proteome profiling between sample groups was assessed by two-dimensional electrophoresis (2-DE) coupled to tandem mass spectrometry. It was found that protein fragmentation increased markedly after US thermal treatment. Specifically, fragments of the major myofibrillar protein, actin, were abundantly over-represented following US heating. In addition, five unfragmented sarcoplasmic proteins (i.e. fatty acid-binding protein, peroxiredoxin-6, superoxide dismutase, carbonyl reductase and aminoacylase) showed increased abundance in the US sample group. These results suggest candidate biomarkers to monitor proteolysis intensity and proteolysis-independent effects linked to cured ham quality by ultrasound application. SignificanceThe present proteome profiling study of treated dry-cured ham demonstrates the impact of ultrasound action on proteins. Moreover, negative organoleptic effects can be appearing with ultrasound treatment due to proteolysis increase. Therefore, the proteolysis monitoring could help to control these effects. In this regards, our results suggest that actin can be a candidate biomarker to monitor proteolysis intensity.

Dysregulation of Cortisol Metabolism in Equine Pituitary Pars Intermedia Dysfunction.

Equine Cushing disease [pituitary pars intermedia dysfunction (PPID)] is a common condition of older horses, but its pathophysiology is complex and poorly understood. In contrast to pituitary-dependent hyperadrenocorticism in other species, PPID is characterized by elevated plasma ACTH but not elevated plasma cortisol. In this study, we address this paradox and the hypothesis that PPID is a syndrome of ACTH excess in which there is dysregulation of peripheral glucocorticoid metabolism and binding. In 14 horses with PPID compared with 15 healthy controls, we show that in plasma, cortisol levels and cortisol binding to corticosteroid binding globulin were not different; in urine, glucocorticoid and androgen metabolites were increased up to fourfold; in liver, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression was reduced; in perirenal adipose tissue, 11β-HSD1 and carbonyl reductase 1 expression was increased; and tissue cortisol levels were not measurably different. The combination of normal plasma cortisol with markedly enhanced urinary cortisol metabolite excretion and dysregulated tissue-specific steroid-metabolizing enzymes suggests that cortisol clearance is increased in horses with PPID. We infer that the ACTH excess may be compensatory and pituitary pathology and autonomous secretion may be a secondary rather than primary pathology. It is possible that successful therapy in PPID may be targeted either at lowering ACTH or, paradoxically, at reducing cortisol clearance.

Screening for new plant hydroxynitrile lyases

The biotransformation of 2-hydroxyacetophenone to (R)-1-phenyl-1, 2-ethanediol (PED) by NADH-dependent (R)-carbonyl reductase (RCR) from Candida parapsilosis is slow and gives low yields, probably as a result of insufficient cofactors. To improve the biotransformation efficiency of (R)-PED from 2-hydroxyacetophenon, an enzyme-coupling system containing RCR and glucose dehydrogenase (GDH) was constructed to strengthen NADH-recycling pathway in Escherichia coli, in which the Shine-Dalgarno sequence and the aligned spacing sequence were used as linkers between them. The introduction of glucose dehydrogenase had little affects on the cell-growth. The co-expression conditions of RCR and glucose dehydrogenase was optimized to rebalance their catalytic functions. The ratio of kcat/KM for enzyme-coupling system catalyzing 2-HAP and glucose was about 1.0, suggesting the good balance between the functions of RCR and GDH. The rebalanced system gave excellent performance in (R)-PED biotransformation: an optical purity of 99.9% and a yield of 99.9% at optimal conditions: 35 °C and pH 7.0. The introduction of glucose dehydrogenase stimulated increases of 23.8% and 63.8%, in optical purity and yield of (R)-PED, and simultaneously reduced the reaction time two-fold. This work provided a valuable method for efficient chiral alcohol production through protein-expression and biotransformation optimization to rebalance cofactor pathways.

Correction: Carbonyl Reductase 1 Offers a Novel Therapeutic Target to Enhance Leukemia Treatment by Arsenic Trioxide.

Correction: Carbonyl Reductase 1 Offers a Novel Therapeutic Target to Enhance Leukemia Treatment by Arsenic Trioxide.

Understanding substrate specificity and enantioselectivity of carbonyl reductase from Candida parapsilosis ATCC 7330 (CpCR): Experimental and modeling studies

The whole cells of Candida parapsilosis ATCC 7330 are a well-established biocatalyst used for oxidation and reduction of various organic compounds to generate chiral synthons. Recombinantly expressed carbonyl reductase (CpCR) from the same strain reduces aryl α–ketoesters to their respective optically pure alcohols but preferentially reduces aliphatic and aryl aldehydes to primary alcohols. The prochiral substrates viz. aryl α-ketoester [Ethyl-2-oxo-4-phenylbutanoate], aryl ketone [Acetophenone] and aliphatic β-ketoester [Ethyl-4,4,4-trifloro-3-oxo-butanoate] get reduced to (R)-alcohols with CpCR while an aryl ketoaldehyde [2-oxo-2-phenylacetaldehyde] gives the (S)-alcohol. The optimal orientation required for the high conversion and desired enantioselectivity was analyzed by docking the α/β ketoesters, ketoaldehyde and a ketone with a modeled CpCR. Aryl α-ketoester, having the lowest free energy (-8.43 kcal/mol), shows the most favorable binding with CpCR (Interaction Energy = 7.9 kcal/mol). Also, the close proximity of aryl α-ketoester to the cofactor NADPH (2.82 Å) facilitates a better Pro-R hydride transfer as compared to other substrates.

Exploring the Metabolism of Loxoprofen in Liver Microsomes: The Role of Cytochrome P450 and UDP-Glucuronosyltransferase in Its Biotransformation.

Loxoprofen, a propionic acid derivative, non-steroidal anti-inflammatory drug (NSAID) is a prodrug that is reduced to its active metabolite, trans-alcohol form (Trans-OH) by carbonyl reductase enzyme in the liver. Previous studies demonstrated the hydroxylation and glucuronidation of loxoprofen. However, the specific enzymes catalyzing its metabolism have yet to be identified. In the present study, we investigated metabolic enzymes, such as cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), which are involved in the metabolism of loxoprofen. Eight microsomal metabolites of loxoprofen were identified, including two alcohol metabolites (M1 and M2), two mono-hydroxylated metabolites (M3 and M4), and four glucuronide conjugates (M5, M6, M7, and M8). Based on the results for the formation of metabolites when incubated in dexamethasone-induced microsomes, incubation with ketoconazole, and human recombinant cDNA-expressed cytochrome P450s, we identified CYP3A4 and CYP3A5 as the major CYP isoforms involved in the hydroxylation of loxoprofen (M3 and M4). Moreover, we identified that UGT2B7 is the major UGT isoform catalyzing the glucuronidation of loxoprofen and its alcoholic metabolites. Further experimental studies should be carried out to determine the potency and toxicity of these identified metabolites of loxoprofen, in order to fully understand of mechanism of loxoprofen toxicity.

Identification and Characterization of a Selective Human Carbonyl Reductase 1 Substrate.

During drug discovery efforts targeting inhibition of cytochrome P450 11B2 (CYP11B2)-mediated production of aldosterone as a therapeutic approach for the treatment of chronic kidney disease and hypertension, (S)-6-(5-fluoro-4-(1-hydroxyethyl)pyridin-3-yl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (1) was identified as a potent and selective inhibitor of CYP11B2. Preclinical studies characterized 1 as low clearance in both in vitro test systems and in vivo in preclinical species. Despite low metabolic conversion, an active ketone metabolite (2) was identified from in vitro metabolite-identification studies. Due to the inhibitory activity of 2 against CYP11B2 as well as the potential for it to undergo reductive metabolism back to 1, the formation and elimination of 2 were characterized and are the focus of this manuscript. A series of in vitro investigations determined that 1 was slowly oxidized to 2 by cytochrome P450s 2D6, 3A4, and 3A5, followed by stereoselective reduction back to 1 and not its enantiomer (3). Importantly, reduction of 2 was mediated by an NADPH-dependent cytosolic enzyme. Studies with human cytosolic fractions from multiple tissues, selective inhibitors, and recombinantly expressed enzymes indicated that carbonyl reductase 1 (CBR1) is responsible for this transformation in humans. Carbonyl reduction is emerging as an important pathway for endogenous and xenobiotic metabolism. With a lack of selective substrates and inhibitors to enable characterization of the involvement of CBR1, 2 could be a useful probe to assess CBR1 activity in vitro in both subcellular fractions and in cell-based systems.

A new role for Carbonyl Reductase 1 on 4-hydroxynonenal detoxification

4-Hydroxy-2-nonenal (HNE) is one of the main products of lipid autoxidation of unsaturated fatty acid (1). It is a highly reactive molecule and has received a particular attention for its biological activity and its role in different diseases (2). HNE metabolism is reported to mainly occur through its conjugation with glutathione and the subsequent formation of 3-glutathionyl-4-hydroxynonanal (GSHNE) (3). This molecule is susceptible to both reductive and oxidative transformations, which occur through the action of either the NADPH-dependent action of aldose reductase or the NADP+-dependent activity of aldehyde dehydrogenase, respectively. In this context it has been identified a role of carbonyl reductase 1 (CBR1) in the detoxification of GSHNE through its oxidation to the corresponding 3-glutathionyl-nonanoic-δ-lactone (4). More recently it has been also reported the capability of the enzyme to reduce GSHNE, together with a number of glutathionylated aldehydes, to the corresponding GS-dihydroxynonane (GSDHN) (5). Thus GSHNE, through its equilibrium between the open aldehyde form and its cyclic hemiacetal acts, with respect to CBR1, as a dual substrate. Being the two red/ox processes linked to the same red/ox cofactor (NADP+/NADPH), it turns out that a CBR1-catalyzed disproportion of GSHNE occurs. The high catalytic efficiency of CBR1 in the GSHNE processing, would suggest this molecule as one of the main physiological substrate of the enzyme. In this contest besides contributing to detoxification processes, CBR1 may be involved in the production of a signaling molecule, GSDHN, which is reported to activate inflammatory processes mediated by NFκB (6). These results could add new relevance on the inhibition of CBR1 activity by specific molecules that could play an anti-inflammatory role.

Expression of engineered carbonyl reductase from Ogataea minuta in Rhodococcus opacus and its application to whole-cell bioconversion in anhydrous solvents

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda etal., J. Biosci. Bioeng., 123, 673-678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24h.

Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L−1 of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L−1 h−1 g−1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase.

Initial characterization of human DHRS1 (SDR19C1), a member of the short-chain dehydrogenase/reductase superfamily

Many enzymes from the short-chain dehydrogenase/reductase superfamily (SDR) have already been well characterized, particularly those that participate in crucial biochemical reactions in the human body (e.g. 11β-hydroxysteroid dehydrogenase 1, 17β-hydroxysteroid dehydrogenase 1 or carbonyl reductase 1). Several other SDR enzymes are completely or almost completely uncharacterized, such as DHRS1 (also known as SDR19C1). Based on our in silico and experimental approaches, DHRS1 is described as a likely monotopic protein that interacts with the membrane of the endoplasmic reticulum. The highest expression level of DHRS1 protein was observed in human liver and adrenals. The recombinant form of DHRS1 was purified using the detergent n-dodecyl-β-D-maltoside, and DHRS1 was proven to be an NADPH-dependent reductase that is able to catalyse the in vitro reductive conversion of some steroids (estrone, androstene-3,17-dione and cortisone), as well as other endogenous substances and xenobiotics. The expression pattern and enzyme activities fit to a role in steroid and/or xenobiotic metabolism; however, more research is needed to fully clarify the exact biological function of DHRS1.

Candidate gene and mechanism investigations in congenital obstructive nephropathy based on bioinformatics analysis.

The aim of the present study was to explore the candidate genes, chemicals and mechanisms of congenital obstructive nephropathy (CON). The gene expression profiles of GSE48041, including 24 kidney tissue samples from megabladder (mgb−/−) mouse were downloaded from the Gene Expression Omnibus database. Samples were divided into 4 groups: Control, mild, moderate and severe. Differentially expressed genes (DEGs), protein-protein interaction network, Kyoto Encyclopedia of Genes and Genomes pathways and transcription factor (TF)-target gene analyses were performed on Set 1 (mild, moderate and severe groups), while Gene Ontology (GO) function enrichment analysis and chemical investigation were performed on Set 2 (severe group). A total of 187 and 139 DEGs were obtained in Set 1 and Set 2, respectively. Chemical carcinogenesis [enriched by genes such as Carbonyl reductase 1 (CBR1)] was one of the most prominent pathways in Set 1. GO analysis for Set 2 revealed that DEGs were mainly assembled in functions such as cellular response to interleukin-1 and cellular response to tumor necrosis. Furthermore, genes such as Fos Proto-Oncogene (FOS) were co-regulated by TFs including RNA polymerase II subunit A (Polr2a) and serum response factor (Srf). Chemical cyclosporine served the most important role in Set 2 by targeting several DEGs in Set 2. DEGs such as CBR1 and FOS, TFs including Polr2a and Srf, and pathways such as chemical carcinogenesis may serve important roles in the process of CON. Interleukin-1 and tumor necrosis function may be novel targets for CON gene therapy. Furthermore, cyclosporine may be a promising option for future CON therapy.

Aldo-keto reductase 1C3 (AKR1C3): a missing piece of the puzzle in the dinaciclib interaction profile

Dinaciclib is a multi-specific cyclin-dependent kinase (CDK) inhibitor with significant preclinical and clinical activity. It inhibits CDK1, CDK2, CDK5, CDK9 and CDK12 in the nanomolar range and exhibits potent antiproliferative effects on various cancers in vitro and in vivo. Aldo-keto reductases (AKR) and carbonyl reductases (CBR) are enzymes involved at the biosynthesis, intermediary metabolism and detoxification processes, but can also play a significant role in cancer resistance. Here, we report that dinaciclib is a strong inhibitor of aldo-keto reductase 1C3 (AKR1C3), an enzyme that is known to be an important regulator of cell proliferation and differentiation. AKR1C3 is overexpressed in a range of cancer types and is also involved in tumour cell resistance to anthracyclines. In our study, dinaciclib displayed tight-binding inhibition of human recombinant AKR1C3 (Kiapp = 0.07µM) and was also active at the cellular level (IC50 = 0.23µM). Dinaciclib acts as a noncompetitive inhibitor with respect to daunorubicin and as an uncompetitive inhibitor with respect to the NADPH. In subsequent experiments, pretreatment with dinaciclib (0.1µM) significantly sensitized AKR1C3-overexpressing anthracycline-resistant cancer cells to daunorubicin. In conclusion, our results indicate that dinaciclib may potentially increase the therapeutic efficacy and safety of anthracyclines by preventing anthracycline resistance and minimizing their adverse effects.

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 arole in oxidative stress defense. Further studies are needed to investigate their potential to serve as novel biomarkers for oxidative stress in Daphnia.

CBR1 rs9024 genotype status impacts the bioactivation of loxoprofen in human liver.

Loxoprofen is an anti-inflammatory drug that requires bioactivation into the trans-OH metabolite to exert pharmacological activity. Evidence suggests that carbonyl reductase 1 (CBR1) is important during the bioactivation of loxoprofen. This study examined the impact of the functional single nucleotide polymorphism CBR1 rs9024 on the bioactivation of loxoprofen in a collection of human liver samples. The synthesis ratios of trans-OH loxoprofen/cis-OH loxoprofen were 33% higher in liver cytosols from donors homozygous for the CBR1 rs9024 G allele in comparison with the ratios in samples from donors with heterozygous GA genotypes. Complementary studies examined the impact of CBR1 rs9024 on the bioactivation of loxoprofen in lymphoblastoid cell lines. CBR1 rs9024 genotype status impacts the synthesis of the bioactive trans-OH metabolite of loxoprofen in human liver.

Expression analysis of CBR3-AS1 and androgen receptor genes in breast cancer

Long non-coding RNAs (lncRNAs) have been shown to participate in the pathogenesis of a variety of cancers including breast cancer. The lncRNA Carbonyl reductase 3-Antisense 1 (CBR3-AS1) has been implicated in some human malignancies in association with androgen receptor (AR). In the present study, we evaluated the expression of CBR3-AS1 and AR transcripts in 52 breast cancer tissues, their corresponding adjacent non-cancerous tissues (ANCTs) and two breast cancer cell lines (MDA-MB-231 and MCF-7). We extracted data of our previously published work regarding expression level of Zinc-finger enhancer binding protein (ZEB1) in the same cohort of patients. CBR3-AS1 was up-regulated in MDA-MB-231 compared with ANCTs. AR was significantly down-regulated in both breast cancer cell lines compared with ANCTs. AR expression was lower in tumoral tissues compared with ANCTs but it did not reach the level of significance. CBR3-AS1 expression has been shown to be significantly elevated in tumor tissues compared with ANCTs (fold change = 4.178, P = 0.021). No significant associations have been detected between the level of transcripts and patients' data except for an association between AR expression and cancer stage (P = 0.037). In luminal B subtype, AR transcript levels were more frequently down-regulated (P = 0.015). A significant correlation has been detected between the levels of CBR3-AS1 and AR transcripts in tumor tissues but not in ANCTs. No significant correlation has been detected between the levels of AR and ZEB1 transcripts in tumor tissues or in ANCTs. The observed significant up-regulation of CBR3-AS1 in breast cancer tissues and cell lines is in line with the suggested oncogenic role of this lncRNA in other cancers.

Semi-rational engineering of carbonyl reductase YueD for efficient biosynthesis of halogenated alcohols with in situ cofactor regeneration

Optically active 1-phenylethanol and its derivatives are versatile chiral precursors for many pharmaceuticals. The increasing market demand of enantiopure alcohols calls for exploration of more robust biocatalysts capable of delivering high conversion rates at high substrate concentrations. The carbonyl reductase YueD from Bacillus subtilis was engineered for improved reduction of halogenated acetophenones. Based on in silico docking and Alanine screening, mutant Val181Ala with 97% conversion of the model substrate 3-bromoacetophenone was obtained. Further molecular simulation analysis suggested expansion of the catalytic pocket and altered substrate orientation to provide a structural basis for the improved activity upon Val181 mutation. To eliminate the requirement of expensive exogenous NADPH and thus afford economical synthesis of enantiopure alcohol products, a cofactor regeneration system based on glucose dehydrogenase was introduced. Since the product of glucose oxidation seemed to inhibit the activity of YueD, xylose was used instead as the co-substrate, which efficiently regenerated NADPH in situ, delivering (S) or (R) alcohols with >99% ee at high conversion rates of 85–99% in the asymmetric reduction of halogenated acetophenones (500 mM). These data demonstrate the industrial potential of the YueD mutant Val181Ala in biosynthesis of valuable chiral alcohols.