RESUMO
Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.
Assuntos
Oxirredutases do Álcool , Etanol , Ticagrelor , Estabilidade Enzimática , Oxirredutases do Álcool/genética , Temperatura , ComputadoresRESUMO
Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCRM3 , coevolved thermostability, and activity. Compared with LsCRM3 , the catalytic efficiency kcat /KM of LsCRM3 -E145A (LsCRM4 ) was increased from 6.6 to 21.9 s-1 mM-1 . Moreover, E145A prolonged the half-life t1/2 at 40°C from 4.1 to 117 h, T m ${T}_{m}$ was increased by 5°C, T 50 30 ${T}_{50}^{30}$ was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% eeP , with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT , the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.
Assuntos
Mutação Puntual , Engenharia de Proteínas , Catálise , Etanol , Especificidade por SubstratoRESUMO
Cytosolic phospholipase A2α (cPLA2α), the key enzyme of the arachidonic acid cascade, is considered to be an interesting target for the development of new anti-inflammatory drugs. Potent inhibitors of the enzyme include indole-5-carboxylic acids with propan-2-one residues in position 1 of the indole. Previously, it was found that central pharmacophoric elements of these compounds are their ketone and carboxylic acid groups, which unfortunately are subject to pronounced metabolism by carbonyl reductases and glucuronosyltransferases, respectively. Here we show that the metabolic stability of these inhibitors can be improved by introducing alkyl substituents in the vicinity of the ketone group or by increasing their rigidity. Furthermore, permeability tests with Caco-2 cells revealed that the indole derivatives have only low permeability, which can be attributed to their affinity to efflux transporters. Among other things, the polar ketone group in the center of the molecules seems to be a decisive factor for their reverse transport. After its removal, the permeability increased significantly. The enhancement in metabolic stability and permeability achieved by the structural variations carried out was accompanied by a more or less pronounced decrease in the inhibitory potency of the compounds against cPLA2α.
Assuntos
Fosfolipases A2 do Grupo IV , Indóis , Humanos , Relação Estrutura-Atividade , Fosfolipases A2 do Grupo IV/metabolismo , Células CACO-2 , Indóis/química , Cetonas/química , Inibidores Enzimáticos/químicaRESUMO
Vibegron is a novel, potent, highly selective ß3-adrenergic receptor agonist for the treatment of overactive bladder with higher therapeutic capacity and lower side effects. Methyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropanoate ((2S,3R)-aminohydroxy ester) is a key chiral intermediate for the synthesis of Vibegron. A novel carbonyl reductase from Exiguobacterium sp. s126 (EaSDR6) was isolated using data mining technology from GenBank database with preferable catalytic activity. Hydrogen bond network regulation was performed using site-directed saturation mutagenesis and combination mutagenesis. The mutant EaSDR6A138L/S193A was obtained with the activity improvement by 4.58 folds compared with the wild type EaSDR6. The Km of EaSDR6A138L/S193A was decreased from 1.57 mM to 0.67 mM, kcat was increased by 2.17 folds, and the overall catalytic efficiency kcat/Km was increased by 5.07 folds. The organic-aqueous biphasic bioreaction system for the asymmetric synthesis of (2S,3R)-aminohydroxy ester was constructed for the first time. Under the substrate concentration of 150 g/L, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99%, and the spatiotemporal yield was 1.55 g/(L·h·g DCW) after 12 h reaction. While the substrate concentration was increased to 200 g/L and the reaction lasted for 36 h, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99% and the spatiotemporal yield was 1.05 g/(L·h·g DCW). The substrate concentration and spatiotemporal yield were higher than ever reported.
Assuntos
Oxirredutases do Álcool , Pirimidinonas , Ligação de Hidrogênio , Oxirredutases do Álcool/genética , ÉsteresRESUMO
The superfamily of short-chain dehydrogenases/reductases (SDRs) is crucial in biosynthetic and signalling pathways, in which the carbonyl reductases (CBRs) subfamily is important in the biosynthesis of tetrahydrobiopterin (BH4). BH4 is an essential coenzyme for animals, and its deficiency can lead to neurological diseases. There are few reports on CBRs involved in BH4 synthesis of silkworms, Bombyx mori. Here, we identified 67 SDR genes in B. mori (BmSDR) through whole genome survey for the first time. Based on bioinformatics analyses and KEGG verification, four BmCBRs that may be related to BH4 synthesis were further characterized and functionally analysed. The results showed these four genes were high expressed in the head and gonads of ah09 (a lem mutant with defective BH4 synthesis). Enzyme activity, BH4 content and the related gene expression levels after intracellular interference with BmCBR and the main catalytic enzymes sepiapterin reductase of B. mori (BmSpr) in the de novo pathway of BH4 showed BmCBR2 plays a role in the salvage pathway. BmCBR3 and BmCBR4 regulate BH4 synthesis through the alternative pathway. Among the four pathways of silkworm BH4 synthesis, the de novo pathway occupies the dominant position, followed by the alternative pathway and salvage pathway. According to the overexpression of BmCBR3 after interference with BmSpr, the BH4 content did not change significantly. It is speculated that BmCBR3 is located upstream of BmSpr. These results provide a theoretical basis for in-depth exploration of the role of BmSDR in B. mori and also provide clues for the research of other animal-related diseases.
Assuntos
Bombyx , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Bombyx/metabolismoRESUMO
Carbonyl reductase from Leifsonia xyli (LXCAR, UniProtKB - T2FLN4) can stereoselectively catalyze the reduction of 3,5-bis(trifluoromethyl)acetophenone (BTAP) to its corresponding alcohol, (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol ((R)-BTPE), which is a valuable chiral intermediate for the synthesis of antiemetic drugs, Aprepitant and Fosaprepitant. Moreover, this protein was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters. Even though molecular modelling of this protein was done by Wang et al. (2014), a crystal structure has not yet obtained. In this study, a single mutant, S154Y, which was shown to have higher catalytic activity toward BTAP than that of the wild type, was overexpressed in Escherichia coli BL21 (DE3), purified, and crystallized. The crystal structure of LXCAR-S154Y explains how the mutant enzyme can work with BTAP more efficiently than wild type carbonyl reductase. Furthermore, the structure explains why LXCAR-S154Y can use either NADH or NADPH efficiently as a cofactor, as well as elucidates a proton relay system present in the enzyme.
Assuntos
Actinobacteria , Oxirredutases do Álcool , Acetofenonas , Oxirredutases do Álcool/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol , Especificidade por SubstratoRESUMO
Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase LsCR for the synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol ((S)-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a "small and smart" mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant LsCRM3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) LsCR, the catalytic activity of LsCRM3 was increased by 4.7 times, the catalytic efficiency kcat/KM value was increased by 2.9 times, and the half-life t1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% (v/v) isopropanol, LsCRM3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e.) in 11 h with a space-time yield (STY) as high as 809 g/Lâd.
Assuntos
2-Propanol , Etanol , Oxirredutases do Álcool/genética , Catálise , EstereoisomerismoRESUMO
Enzyme stereoselectivity control is still a major challenge. To gain insight into the molecular basis of enzyme stereo-recognition and expand the source of antiPrelog carbonyl reductase toward ß-ketoesters, rational enzyme design aiming at stereoselectivity inversion was performed. The designed variant Q139G switched the enzyme stereoselectivity toward ß-ketoesters from Prelog to antiPrelog, providing corresponding alcohols in high enantiomeric purity (89.1-99.1 % ee). More importantly, the well-known trade-off between stereoselectivity and activity was not found. Q139G exhibited higher catalytic activity than the wildtype enzyme, the enhancement of the catalytic efficiency (kcat /Km ) varied from 1.1- to 27.1-fold. Interestingly, the mutant Q139G did not lead to reversed stereoselectivity toward aromatic ketones. Analysis of enzyme-substrate complexes showed that the structural flexibility of ß-ketoesters and a newly formed cave together facilitated the formation of the antiPrelog-preferred conformation. In contrast, the relatively large and rigid structure of the aromatic ketones prevents them from forming the antiPrelog-preferred conformation.
Assuntos
Oxirredutases do Álcool , Álcoois , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Cetonas , Estereoisomerismo , Especificidade por SubstratoRESUMO
Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (kcat /KM = 47.3 s-1 ·mM-1 ), improved stereoselectivity (from moderate selectivity (deP = 66.7%) to strict (S)-selectivity (deP > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.
Assuntos
Oxirredutases do Álcool , Domínio Catalítico/genética , Engenharia de Proteínas/métodos , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Biocatálise , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/enzimologia , Kluyveromyces/genética , Simulação de Acoplamento Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: The aim of this case-control study was to document maternal, umbilical arterial metabolic levels and correlations in pregnancies with and without 25-hydroxyvitamin D [25(OH)D] deficiency, while, also investigating the expression of nuclear factor erythroid 2 related factor 2 (Nrf2) and carbonyl reductase 1 (CBR1) in the placenta. METHODS: One hundred participants, 50 deficient for 25(OH)D and 50 normal, were recruited from among hospitalized single-term pregnant women who had elected for cesarean section. Umbilical arterial and placental samples were collected during cesarean section. Metabolic levels were assessed for the 25(OH)D deficiency and control groups' maternal, umbilical arterial samples. Nrf2 and CBR1 expression levels were investigated in the placentas of 12 pregnant women with 25(OH)D deficiency and 12 controls. RESULTS: Compared with the control participants, the 25(OH)D deficient women had significantly higher triglyceride (TG) levels (3.80 ± 2.11 vs. 2.93 ± 1.16 mmol/L, 3.64 ± 1.84 vs. 2.81 ± 1.16 mmol/L, p < .01, .001); lower high density lipoprotein cholesterol (HDL-C) levels (1.54 ± 0.32 vs. 1.82 ± 0.63 mmol/L, 1.41 ± 0.72 vs. 2.44 ± 1.68 mmol/L, p < .001, .01) in both material blood and the umbilical artery. In addition, Nrf2 and CBR1 expression levels were lower in the maternal 25(OH)D deficient placenta. CONCLUSION: 25(OH)D deficient pregnant women have higher TG levels and lower HDL-C levels in both material blood and the umbilical artery. TG level is negatively correlated with 25(OH)D in both the maternal serum and infant umbilical artery. 25(OH)D deficiency also lowers placental expression of Nrf2 and CBR1.Supplemental data for this article is available online at here.
Assuntos
Oxirredutases do Álcool/análise , Fator 2 Relacionado a NF-E2/análise , Placenta/química , Complicações na Gravidez/metabolismo , Deficiência de Vitamina D/complicações , Vitamina D/análogos & derivados , Adulto , Glicemia/análise , Estudos de Casos e Controles , HDL-Colesterol/sangue , Feminino , Humanos , Gravidez , Estudos Retrospectivos , Triglicerídeos/sangue , Artérias Umbilicais , Vitamina D/sangue , Deficiência de Vitamina D/metabolismoRESUMO
A strain NQ1, which showed efficient asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to enantiopure (S)-[3,5-bis(trifluoromethyl)phenyl]ethanol ((S)-BTPE), which is the key intermediate for the synthesis of a receptor antagonist and antidepressant, was isolated from a soil sample. Based on its morphological and internal transcribed spacer sequence, the strain NQ1 was identified to be Rhodotorula mucilaginosa NQ1. Some key reaction parameters involved in the bioreduction catalyzed by whole cells of R. mucilaginosa NQ1 were subsequently optimized, and the optimized conditions for the synthesis of (S)-BTPE were determined to be as follows: 5·0 ml phosphate buffer (200 mmol l-1 , pH 7·0), 80 mmol l-1 of BTAP, 250 g (wet weight) l-1 of resting cell, 35 g l-1 of glucose and a reaction for 18 h at 30°C and 180 rev min-1 . The strain NQ1 exhibited a best yield of 99% and an excellent enantiomeric excess of 99% for the preparation of (S)-BTPE under the above optimal conditions, and could also asymmetrically reduce a variety of bulky prochiral carbonyl compounds to their corresponding optical hydroxyl compound with excellent enantioselectivity. These results indicated that R. mucilaginosa NQ1 had a good capacity to reduce BTAP to its corresponding (S)-BTPE, and might be a new potential biocatalyst for the production of valuable chiral hydroxyl compounds in industry.
Assuntos
Aprepitanto/metabolismo , Reatores Biológicos/microbiologia , Rhodotorula/classificação , Rhodotorula/metabolismo , Acetofenonas/química , Oxirredução , Álcool Feniletílico/química , Rhodotorula/isolamento & purificaçãoRESUMO
Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed.
Assuntos
Oxirredutases do Álcool/metabolismo , NADP/metabolismo , Oxirredutases do Álcool/química , Domínio Catalítico , Glutationa/análogos & derivados , Glutationa/metabolismo , Humanos , Especificidade por SubstratoRESUMO
BACKGROUND: (S)-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines. (S)-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP+ to NADPH, while endo-ß-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-ß-1,4-xylanase 2 were introduced into the (S)-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. RESULTS: We constructed several coupled multi-enzyme systems by introducing (S)-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-ß-1,4-xylanase 2 into Escherichia coli. Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli/pET-G-S-2 expressed all three enzymes, and this strain produced (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% (6 g/L 2-HAP) under optimal conditions of 35 °C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-ß-1,4-xylanase 2 into the (S)-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 to 28 h. CONCLUSIONS: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.
Assuntos
Escherichia coli/metabolismo , Etilenoglicol/metabolismo , Xilanos/metabolismoRESUMO
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.
Assuntos
Oxirredutases do Álcool/biossíntese , Bombas (Dispositivos Explosivos) , Monitoramento Ambiental , Substâncias Explosivas/toxicidade , Resíduos Perigosos , Mytilus edulis/efeitos dos fármacos , Trinitrotolueno/toxicidade , Poluentes Químicos da Água/toxicidade , Oxirredutases do Álcool/genética , Animais , Biologia Computacional , Relação Dose-Resposta a Droga , Biomarcadores Ambientais/genética , Indução Enzimática , Mytilus edulis/enzimologia , Mytilus edulis/genética , Oceanos e Mares , Medição de Risco , II Guerra MundialRESUMO
tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate [(3R,5S)-CDHH] is the key chiral intermediate to synthesize the side chain of the lipid-lowering drug rosuvastatin. Carbonyl reductases showed excellent activity for the biosynthesis of (3R,5S)-CDHH. The requirement of cofactor NADH/NADPH leads to high cost for the industrial application of carbonyl reductases. In this study, a self-sufficient biocatalyst based on carbonyl reductase and NADP+ co-immobilization strategy was developed on an amino resin carrier LX-1000HAA (SCR-NADP+@LX-1000HAA). The self-sufficient biocatalyst achieved in situ cofactor regeneration and showed the activity recovery of 77.93% and the specific activity of 70.45 U/g. Asymmetric synthesis of (3R,5S)-CDHH using SCR-NADP+@LX-1000HAA showed high enantioselectivity (> 99% e.e.) and yield (98.54%). Batch reactions were performed for ten cycles without extra addition of NADP+, and the total yield of (3R,5S)-CDHH achieved at 10.56 g/g biocatalyst. The present work demonstrated the potential of the self-sufficient biocatalyst for the asymmetric biosynthesis of rosuvastatin intermediate.
Assuntos
Oxirredutases do Álcool/química , Biocatálise , Candida/enzimologia , Caproatos/síntese química , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , NADP/química , Oxirredutases do Álcool/genética , Candida/genética , Caproatos/química , Enzimas Imobilizadas/genética , Proteínas Fúngicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rosuvastatina Cálcica/síntese química , Rosuvastatina Cálcica/químicaRESUMO
The plant kingdom supports an extraordinary chemical diversity, with terpenoids representing a particularly diversified class of secondary (or specialized) metabolites. Volatile and semi-volatile terpenoids in the C10-C20 range are often formed in specialized cell types and secretory structures. In the angiosperm lineage, glandular trichomes play an important role in enabling the biosynthesis and storage (or in some cases secretion) of functionalized terpenoids. The 'decoration' of a terpenoid scaffold with functional groups changes its physical and chemical properties, and can therefore affect the perception of a specific metabolite by other organisms. Because of the ecological implications (e.g. plant-herbivore interactions) and commercial relevance (e.g. volatiles used in the flavor and fragrance industries), terpenoid functionalization has been researched extensively. Recent successes in the cloning and functional evaluation of genes as well as the structural and biochemical characterization of enzyme catalysts have laid the foundation for an improved understanding of how pathways toward functionalized monoterpenes may have evolved. In this review, we will focus on an up-to-date account of functionalization reactions present in glandular trichomes.
Assuntos
Magnoliopsida/genética , Monoterpenos/metabolismo , Tricomas/enzimologia , Regulação da Expressão Gênica de Plantas , Magnoliopsida/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
BACKGROUND: Saccharomyces cerevisiae AN120 osw2∆ spores were used as a host with good resistance to unfavorable environment. This work was undertaken to develop a new yeast spore-encapsulation of Candida parapsilosis Glu228Ser/(S)-carbonyl reductase II and Bacillus sp. YX-1 glucose dehydrogenase for efficient chiral synthesis in organic solvents. RESULTS: The spore microencapsulation of E228S/SCR II and GDH in S. cerevisiae AN120 osw2∆ catalyzed (R)-phenylethanol in a good yield with an excellent enantioselectivity (up to 99%) within 4 h. It presented good resistance and catalytic functions under extreme temperature and pH conditions. The encapsulation produced several chiral products with over 70% yield and over 99% enantioselectivity in ethyl acetate after being recycled for 4-6 times. It increased substrate concentration over threefold and reduced the reaction time two to threefolds compared to the recombinant Escherichia coli containing E228S and glucose dehydrogenase. CONCLUSIONS: This work first described sustainable enantioselective synthesis without exogenous cofactors in organic solvents using yeast spore-microencapsulation of coupled alcohol dehydrogenases.
Assuntos
Oxirredutases do Álcool/metabolismo , Bacillus/metabolismo , Candida parapsilosis/metabolismo , Composição de Medicamentos/métodos , Glucose 1-Desidrogenase/metabolismo , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/metabolismo , SolventesRESUMO
The current authors previously reported that a carbonyl reductase 1 (CR1) DNA-dendrimer complex could potentially be used in gene therapy for peritoneal metastasis of ovarian cancer. The aims of the current study were to observe the cellular dynamics of peritoneal metastasis of epithelial ovarian cancer cells and to ascertain changes in the dynamics of ovarian cancer cells as a result of transfection of CR1 DNA. (1) Artificial human peritoneal tissue (AHPT) was seeded with serous ovarian cancer cells, and the process leading to development of peritoneal carcinomatosis was observed over time. (2) Peritoneal carcinomatosis was produced in mice and compared to a model using AHPT to determine the appropriateness of AHPT. (3) CR1 DNA was transfected into cancer cells seeded on AHPT, and the dynamics of cancer cells were observed over time. (1) Cancer cells perforated the mesothelium, leaving normal mesothelium intact. However, the cells proliferated between the layers of the mesothelium, forming a mass. After 24 h, cancer cells had invaded the lymphatics, and after 48-72 h cancer cells had invaded deep into the mesothelium, where they formed a mass. (2) Invasion of the peritoneum by cancer cells in a murine model of peritoneal carcinomatosis resembled that in a model using AHPT, and results substantiated the reproducibility of peritoneal carcinomatosis in AHPT. (3) Proliferation of cells transfected with CR1 DNA was significantly inhibited on AHPT, and necrosis was evident. Nevertheless, cancer cell invasion deep into the mesothelium was not inhibited. Use of a new tool, AHPT, in an in vitro model of peritoneal metastasis revealed that CR1 DNA inhibited cancer cell proliferation. CR1 DNA does not play a role in inhibiting invasion of the mesothelium during peritoneal metastasis, but it does affect cancer cell proliferation. Results suggested that CR1 DNA inhibits cancer cell proliferation via necrosis.
Assuntos
Oxirredutases do Álcool/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Oxirredutases do Álcool/genética , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Expressão Gênica , Genes Reporter , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/ultraestrutura , Neoplasias Peritoneais/ultraestruturaRESUMO
AIMS: We aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis. METHODS AND RESULTS: 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 6·96 U mg-1 (99·8% e.e.) and 7·85 U mg-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 (20·0 g l-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. CONCLUSIONS: A new whole-cell recombinant E. coli-based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed. SIGNIFICANCE AND IMPACT OF THE STUDY: We proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.
Assuntos
Oxirredutases do Álcool/metabolismo , Álcoois/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Yarrowia/enzimologia , Acetofenonas/química , Acetofenonas/metabolismo , Oxirredutases do Álcool/genética , Álcoois/química , Proteínas de Bactérias/genética , Escherichia coli/química , Escherichia coli/genética , Engenharia Metabólica , Estereoisomerismo , Yarrowia/genéticaRESUMO
A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.