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
Group A rotaviruses cause severe gastroenteritis in infants and young children worldwide, with P[II] genogroup rotaviruses (RVs) responsible for >90% of global cases. RVs have diverse host ranges in different human and animal populations determined by host histo-blood group antigen (HBGA) receptor polymorphism, but details governing diversity, host ranges, and species barriers remain elusive. In this study, crystal structures of complexes of the major P[II] genogroup P[4] and P[8] genotype RV VP8* receptor-binding domains together with Lewis epitope-containing LNDFH I glycans in combination with VP8* receptor-glycan ligand affinity measurements based on NMR titration experiments revealed the structural basis for RV genotype-specific switching between ßß and ßα HBGA receptor-binding sites that determine RV host ranges. The data support the hypothesis that P[II] RV evolution progressed from animals to humans under the selection of type 1 HBGAs guided by stepwise host synthesis of type 1 ABH and Lewis HBGAs. The results help explain disease burden, species barriers, epidemiology, and limited efficacy of current RV vaccines in developing countries. The structural data has the potential to impact the design of future vaccine strategies against RV gastroenteritis.
Assuntos
Antígenos de Grupos Sanguíneos/imunologia , Evolução Molecular , Rotavirus/genética , Cristalografia por Raios X , Especificidade de Hospedeiro/genética , Humanos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Rotavirus/química , Rotavirus/imunologia , Proteínas não Estruturais Virais/química , Vacinas Virais/imunologiaRESUMO
Aldo-keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH-dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t-butyl 6-chloro (5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) were improved by mutating its residues in the loop regions around the substrate-binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67-fold higher catalytic efficiency compared to the wild-type (WT) KdAKR, and improved R-selectivity toward (5S)-CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3-fold increase in half-life (t1/2 ) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)-CHOH to diastereomeric pure t-butyl 6-chloro-(3R, 5S)-dihydroxyhexanoate ((3R, 5S)-CDHH) within 8.0 h, with a space-time yield of 300.7 g/L/day. Our results deepen the understandings of the structure-function relationship of AKRs, providing a certain guidance for the modification of other AKRs.
Assuntos
Caproatos , Kluyveromyces , Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/química , Catálise , Aldeído Redutase/genéticaRESUMO
Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidation of the structure-function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information.
Assuntos
Aminoácidos , Engenharia de Proteínas , Biocatálise , Catálise , Estabilidade EnzimáticaRESUMO
Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a ß-sheet, referred as the "ßα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ß-sheets, referred to as the "ßß binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ßα binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.
Assuntos
Proteínas do Capsídeo/química , Antígenos do Grupo Sanguíneo de Lewis/química , Simulação de Acoplamento Molecular , Rotavirus/química , Proteínas do Capsídeo/metabolismo , Células HT29 , Humanos , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Rotavirus/metabolismoRESUMO
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
Sepsis-associated acute kidney injury (SA-AKI) is a significant problem in the critically ill that causes increased death. Emerging understanding of this disease implicates metabolic dysfunction in its pathophysiology. This study sought to identify specific metabolic pathways amenable to potential therapeutic intervention. Using a murine model of sepsis, blood and tissue samples were collected for assessment of systemic inflammation, kidney function, and renal injury. Nuclear magnetic resonance (NMR)-based metabolomics quantified dozens of metabolites in serum and urine that were subsequently submitted to pathway analysis. Kidney tissue gene expression analysis confirmed the implicated pathways. Septic mice had elevated circulating levels of inflammatory cytokines and increased levels of blood urea nitrogen and creatinine, indicating both systemic inflammation and poor kidney function. Renal tissue showed only mild histological evidence of injury in sepsis. NMR metabolomic analysis identified the involvement of mitochondrial pathways associated with branched-chain amino acid metabolism, fatty acid oxidation, and de novo NAD+ biosynthesis in SA-AKI. Renal cortical gene expression of enzymes associated with those pathways was predominantly suppressed. Renal cortical fatty acid oxidation rates were lower in septic mice with high inflammation, and this correlated with higher serum creatinine levels. Similar to humans, septic mice demonstrated renal dysfunction without significant tissue disruption, pointing to metabolic derangement as an important contributor to SA-AKI pathophysiology. Metabolism of branched-chain amino acid and fatty acids and NAD+ synthesis, which all center on mitochondrial function, appeared to be suppressed. Developing interventions to activate these pathways may provide new therapeutic opportunities for SA-AKI.NEW & NOTEWORTHY NMR-based metabolomics revealed disruptions in branched-chain amino acid metabolism, fatty acid oxidation, and NAD+ synthesis in sepsis-associated acute kidney injury. These pathways represent essential processes for energy provision in renal tubular epithelial cells and may represent targetable mechanisms for therapeutic intervention.
Assuntos
Injúria Renal Aguda/sangue , Injúria Renal Aguda/urina , Imageamento por Ressonância Magnética/métodos , Metabolômica/métodos , Mitocôndrias/metabolismo , Sepse/complicações , Animais , Biomarcadores/sangue , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Inflamação/sangue , Inflamação/metabolismo , Inflamação/urina , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Enzyme engineering usually generates trade-offs between activity, stability, and selectivity. Herein, we report semirational engineering of an aldo-keto reductase (AKR) KmAKR for simultaneously enhancing its thermostability and catalytic activity. Previously, we constructed KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C), which showed outstanding activity towards t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), and t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, the key chiral building blocks of rosuvastatin and atorvastatin. Under the guidance of computer-aided design including consensus residues analysis and molecular dynamics (MD) simulations, K164, S182, S232, and Q266 were dug out for their thermostability conferring roles, generating the "best" mutant KmAKRM13 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The Tm and T5015 values of KmAKRM13 were 10.4 and 6.1°C higher than that of KmAKRM9 , respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over KmAKRM9 . Structural analysis indicated that stabilization of the α-helixes mainly contributed to thermostability enhancement. Under the optimized conditions, KmAKRM13 completely asymmetrically reduced 400 g/l t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) in 8.0 h at a high substrate to catalyst ratio (S/C) of 106.7 g/g, giving diastereomerically pure (3R,5S)-CDHH (>99.5% d.e.P ) with a space-time yield (STY) of 449.2 g/l·d.
Assuntos
Aldo-Ceto Redutases/química , Candida parapsilosis/enzimologia , Proteínas Fúngicas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Aldo-Ceto Redutases/genética , Candida parapsilosis/genética , Proteínas Fúngicas/genéticaRESUMO
Xylanase is efficient for xylan degradation and widely applied in industries. We found a GH11 family xylanase (Xyn11A) with high thermostability and catalytic activity from compost metatranscriptome. This xylanase has the optimal reaction temperature at 80 °C with the activity of 2907.3 U/mg. The X-ray crystallographic structure shows a typical "right hand" architecture, which is the characteristics of the GH11 family enzymes. Comparing it with the mesophilic XYN II, a well-studied GH11 xylanase from Trichoderma reesei, Xyn11A is more compact with more H-bonds. Our mutagenic results show that the electrostatic interactions in the thumb and palm region of Xyn11A could result in its high thermostability and activity. Introducing a disulfide bond at the N-terminus further increased its optimal reaction temperature to 90 °C with augmented activity. KEY POINTS: ⢠A hyperthermophilic xylanase with high activity was discovered using the metatranscriptomic method. ⢠The mechanisms of thermophilicity and high activity were revealed using X-ray crystallography, mutagenesis, and molecular dynamics simulations. ⢠The thermostability and activity were further improved by introducing a disulfide bond.
Assuntos
Compostagem , Endo-1,4-beta-Xilanases , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , HypocrealesRESUMO
Pentapeptide repeat proteins (PRPs) represent a large superfamily with more than 38 000 sequences in nearly 3500 species, the majority belonging to cyanobacteria but represented among all branches of life. PRPs contain at least eight consecutive pentapeptide repeats with the consensus (A/C/S/V/T/L/I)(D/N/S/K/E/I/R)(L/F)(S/T/R/E/Q/K/V/D)(G/D/E/N/R/Q/K). PRPs fold into right-handed quadrilateral ß helices, also known as repeat-five-residue (Rfr)-folds, with four consecutive pentapeptide repeats comprising a single coil, the ~90° change in polypeptide direction in square-shaped coils achieved by type I, II and IV ß turns, and hydrogen bonds between coils establishing ß ladders on each Rfr-fold face. PRPs are broadly categorized into group 1 and 2 involved in antibiotic resistance and group 3 currently having unknown functions. Motivated by their intriguing structures, we are investigating PRP biophysical characteristics, including Rfr-fold thermal stability, ß turn and ß ladder hydrogen bond amide exchange rates and backbone dynamics. Here, we present analysis of 20 ns molecular dynamics (MD) simulations and all atom normal mode analysis (aaNMA) calculations for four group 1 and group 2 and four group 3 PRPs whose structures have been determined by X-ray crystallography. The MD cross-correlation matrices and aaNMA indicated strong correlated motion between adjacent coils and weak coupled motion between coils separated by one or more intervening coils. Slow anticorrelated motions were detected between adjacent coils in aaNMA modes that we hypothesize are requisite to access exchange-competent states necessary to permit solvent exchange of amide hydrogens involved in ß-ladder and ß-turns hydrogen bonds, which can have lifetimes on the order of months.
Assuntos
Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Dobramento de Proteína , Animais , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cianobactérias/química , Cianobactérias/metabolismo , Medição da Troca de Deutério , Humanos , Ligação de Hidrogênio , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Sequências Repetitivas de Aminoácidos , TermodinâmicaRESUMO
At present, many Deep Neural Network (DNN) methods have been widely used for hyperspectral image classification. Promising classification results have been obtained by utilizing such models. However, due to the complexity and depth of the model, increasing the number of model parameters may lead to an overfitting of the model, especially when training data are insufficient. As the performance of the model mainly depends on sufficient data and a large network with reasonably optimized hyperparameters, using DNNs for classification requires better hardware conditions and sufficient training time. This paper proposes a feature fusion and multi-layered gradient boosting decision tree model (FF-DT) for hyperspectral image classification. First, we fuse extended morphology profiles (EMPs), linear multi-scale spatial characteristics, and nonlinear multi-scale spatial characteristics as final features to extract both special and spectral features. Furthermore, a multi-layered gradient boosting decision tree model is constructed for classification. We conduct experiments based on three datasets, which in this paper are referred to as the Pavia University, Indiana Pines, and Salinas datasets. It is shown that the proposed FF-DT achieves better performance in classification accuracy, training conditions, and time consumption than other current classical hyperspectral image classification methods.
RESUMO
Recognition of specific cell surface glycans, mediated by the VP8* domain of the spike protein VP4, is the essential first step in rotavirus (RV) infection. Due to lack of direct structural information of virus-ligand interactions, the molecular basis of ligand-controlled host ranges of the major human RVs (P[8] and P[4]) in P[II] genogroup remains unknown. Here, through characterization of a minor P[II] RV (P[19]) that can infect both animals (pigs) and humans, we made an important advance to fill this knowledge gap by solving the crystal structures of the P[19] VP8* in complex with its ligands. Our data showed that P[19] RVs use a novel binding site that differs from the known ones of other genotypes/genogroups. This binding site is capable of interacting with two types of glycans, the mucin core and type 1 histo-blood group antigens (HBGAs) with a common GlcNAc as the central binding saccharide. The binding site is apparently shared by other P[II] RVs and possibly two genotypes (P[10] and P[12]) in P[I] as shown by their highly conserved GlcNAc-interacting residues. These data provide strong evidence of evolutionary connections among these human and animal RVs, pointing to a common ancestor in P[I] with a possible animal host origin. While the binding properties to GlcNAc-containing saccharides are maintained, changes in binding to additional residues, such as those in the polymorphic type 1 HBGAs may occur in the course of RV evolution, explaining the complex P[II] genogroup that mainly causes diseases in humans but also in some animals.
Assuntos
Especificidade de Hospedeiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Infecções por Rotavirus/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Zoonoses/virologia , Animais , Sítios de Ligação , Variação Genética , Genótipo , Humanos , Polissacarídeos/metabolismo , Rotavirus/química , Rotavirus/genéticaRESUMO
At2g44920 from Arabidopsis thaliana is a pentapeptide-repeat protein (PRP) composed of 25 repeats capped by N- and C-terminal α-helices. PRP structures are dominated by four-sided right-handed ß-helices typically consisting of mixtures of type II and type IV ß-turns. PRPs adopt repeated five-residue (Rfr) folds with an Rfr consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Unlike other PRPs, At2g44920 consists exclusively of type II ß-turns. At2g44920 is predicted to be located in the thylakoid lumen although its biochemical function remains unknown. Given its unusual structure, we investigated the biophysical properties of At2g44920 as a representative of the ß-helix family to determine if it had exceptional global stability, backbone dynamics, or amide hydrogen exchange rates. Circular dichroism measurements yielded a melting point of 62.8°C, indicating unexceptional global thermal stability. Nuclear spin relaxation measurements indicated that the Rfr-fold core was rigid with order parameters ranging from 0.7 to 0.9. At2g44920 exhibited a striking range of amide hydrogen exchange rates spanning 10 orders of magnitude, with lifetimes ranging from minutes to several months. A weak correlation was found among hydrogen exchange rates, hydrogen bonding energies, and amino acid solvent-accessible areas. Analysis of contributions from fast (approximately picosecond to nanosecond) backbone dynamics to amide hydrogen exchange rates revealed that the average order parameter of amides undergoing fast exchange was significantly smaller compared to those undergoing slow exchange. Importantly, the activation energies for amide hydrogen exchange were found to be generally higher for the slowest exchanging amides in the central Rfr coil and decreased toward the terminal coils. This could be explained by assuming that the concerted motions of two preceding or following coils required for hydrogen bond disruption and amide hydrogen exchange have a higher activation energy compared to that required for displacement of a single coil to facilitate amide hydrogen exchange in either the terminal or penultimate coils.
Assuntos
Amidas/metabolismo , Proteínas de Arabidopsis/metabolismo , Hidrogênio/metabolismo , Amidas/química , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dicroísmo Circular , Escherichia coli , Congelamento , Hidrogênio/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
The aldo-keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer-aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6 . Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The "best" mutant KdAKRM10 (KdAKRM6 -S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T50 15 and Tm values were increased by 10.2 and 10.0°C, respectively, and half-life (t1/2 ) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6 . Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10 , thus improving its stability. The results validated the feasibility of the computer-aided design strategy in improving the stability of AKRs.
Assuntos
Aldeído Redutase , Caproatos , Aldo-Ceto Redutases/química , Aldo-Ceto Redutases/genética , Caproatos/químicaRESUMO
Pullulanase is a well-known debranching enzyme that can specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four residues (A365, V401, H499, and T504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase (PulAR) were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Five beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 â, pH 6.0 and 5.0, respectively. In addition, its thermostabilities at 60 â and 65 â were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.
RESUMO
(S)-2-chlorophenylglycine ((S)-CPG) is a key chiral intermediate for the synthesis of clopidogrel. Herein, a novel, efficient and environmentally friendly chemo-enzymatic route for the preparation of optically pure (S)-CPG was developed. A straightforward chemical synthesis of the corresponding prochiral keto acid substrate (2-chlorophenyl)glyoxylic acid (CPGA) was developed with 91.7% yield, which was enantioselectively aminated by leucine dehydrogenase (LeuDH) to (S)-CPG. Moreover, protein engineering of LeuDH was performed via directed evolution and semi-rational design. A beneficial variant EsLeuDH-F362L with enlarged substrate-binding pocket and increased hydrogen bond between K77 and substrate CPGA was constructed, which exhibited 2.1-fold enhanced specific activity but decreased thermal stability. Coupled with a glucose dehydrogenase from Bacillus megaterium (BmGDH) for NADH regeneration, EsLeuDH-F362L completely converted up to 0.5 M CPGA to (S)-CPG in 8 h at 40 °C.
Assuntos
Proteínas de Bactérias , NAD , Proteínas de Bactérias/metabolismo , Biocatálise , Clopidogrel , Glucose 1-Desidrogenase/metabolismo , Leucina Desidrogenase/metabolismo , NAD/metabolismoRESUMO
ß-1,3-glucanase can specifically hydrolyze glucans to oligosaccharides and has potential applications in biotechnology. We used the metatranscriptomic technology to discover a thermophilic ß-1,3-glucanase from compost. The phylogenetic study shows that it belongs to the family 16 glycoside hydrolase (GH16) and is most homologous with an enzyme from Streptomyces sioyaensis, an actinobacterium. It has the activity of 146.9 U/mg in the optimal reaction condition (75 °C and pH 5.5). Its catalytic domain was crystallized and diffracted to 1.14 Å resolution. The crystal structure shows a sandwich-like ß-jelly-roll fold with two disulfide bonds. After analyzing the occurring frequencies of these cysteine residues, we designed two mutants (C160G and C180I) to study the role of these disulfide bonds. Both mutants have decreased their optimal temperature from 75 to 70 °C, which indicate that the disulfide bonds are important to maintain thermostability. Interestingly, the activity of C160G has increased ~ 17% to reach 171.4 U/mg. We speculate that the increased activity of C160G mutant is due to increased dynamics near the active site. Our studies give a good example of balancing the rigidity and flexibility for enzyme activity, which is helpful for protein engineering.
RESUMO
BACKGROUND: Aldo-keto reductases-catalyzed transformations of ketones to chiral alcohols have become an established biocatalytic process step in the pharmaceutical industry. Previously, we have discovered an aldo-keto reductase (AKR) from Kluyveromyces marxianus that is active to the aliphatic tert-butyl 6-substituted (5R/S)-hydroxy-3-oxohexanoates, but it is inactive to aromatic ketones. In order to acquire an excellent KmAKRmutant for ensuring the simultaneous improvement of activity-thermostability toward tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) and broadening the universal application prospects toward more substrates covering both aliphatic and aromatic ketones, a fluorescence-based high-throughput (HT) screening technique was established. MAIN METHODS AND MAJOR RESULTS: The directed evolution of KmAKR variant M5 (KmAKR-W297H/Y296W/K29H/Y28A/T63M) produced the "best" variant M5-Q213A/T23V. It exhibited enhanced activity-thermostability toward (5R)-1, improved activity toward all 18 test substrates and strict R-stereoselectivity toward 10 substrates in comparison to M5. The enhancement of enzymatic activity and the extension of substrate scope covering aromatic ketones are proposed to be largely attributed to pushing the binding pocket of M5-Q213A/T23V to the enzyme surface, decreasing the steric hindrance at the entrance and enhancing the flexibility of loops surrounding the active center. In addition, combined with 0.94 g dry cell weight (DCW) L-1 glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration, 2.81 g DCW L-1 M5-Q213A/T23V completely converted (5R)-1 of up to 450 g L-1 at 120 g g-1 substrates/catalysts (S/C), yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2, > 99.5% d.e.p ) with a space-time yield (STY) of 1.08 kg L-1 day-1 . CONCLUSIONS: A fluorescence-based HT screening system was developed to tailor KmAKR's activity, thermostability and substrate scope. The "best" variant M5-Q213A/T23V holds great potential application for the synthesis of aliphatic/aromatic R-configuration alcohols.
Assuntos
Kluyveromyces , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases/metabolismo , Catálise , Fluorescência , Especificidade por SubstratoRESUMO
Comparative proteomes of Actinoplanes utahensis ZJB-03852 grown on various saccharides (glucose, maltotriose, maltose, glucose + maltose) were analyzed using 2D-DIGE and MALDI-TOF/TOF-MS. Acarbose was detected in all groups except in the glucose only culture. The abundance of acarbose synthesis proteins AcbV, AcbK, AcbL and AcbN was highest in the medium containing mixed glucose and maltose. The accumulation of Zwf and Xpk1 in acarbose-producing media indicated that the cyclitol moiety of acarbose was derived from pentose phosphate pathway. The elevation of GlnA supported that glutamine was a good nitrogen source of the nitrogen-atom in acarbose synthesis. SIGNIFICANCE: Non-insulin-dependent diabetes mellitus, also known as Type II diabetes, constitutes >90% of the diabetes mellitus worldwide. Acarbose is clinically utilized to treat Type II diabetes, but the fermentation process of acarbose-producing Actinoplanes is usually accompanied with structural analogues of acarbose. In this study, we compared the proteomics of Actinoplanes utahensis ZJB-03852 grown on various saccharides by 2D-DIGE and MALDI-TOF/TOF-MS. Our findings highlighted the importance of key proteins in the formation of acarbose and its analogues when A. utahensis was cultivated in various saccharides. These results revealed fundamental data to elucidate the complexity of formation of acarbose analogues.
Assuntos
Actinoplanes , Diabetes Mellitus Tipo 2 , Acarbose , Humanos , Proteoma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletroforese em Gel Diferencial BidimensionalRESUMO
Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum (SpeedVac) and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, 40-50 metabolites were identified as having statistically significant differences in abundance in redissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in the lyophilized samples, for example, n-methyl-α-aminoisobutyric acid, n-methylnicotimamide, sarcosine and 3-hydroxyisovaleric acid, whereas some metabolites were only present in SpeedVac dried samples, for example, trimethylamine. This research demonstrates that the choice of drying technique used during the preparation of samples of human cell lines or tissue extracts can significantly influence the observed metabolome, making it important to carefully consider the selection of a drying method prior to preparation of such samples for metabolic profiling.