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1.
Bioresour Technol ; 330: 125022, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33765631

RESUMEN

This work aims to synthesize S-(4-chlorophenyl)-(pyridin-2-yl) methanol (S-CPMA) in a green, economic, and efficient way. In the water-cyclohexane liquid-liquid system, recombinant Escherichia coli (E. coli) was used as a whole-cell catalyst and retained > 60% of its catalytic activity after five reuse cycles. In situ accumulation of the substrate/product in the organic phase effectively improves substrate tolerance and reduces product inhibition and toxicity. Meanwhile, a microreaction system consisting of membrane dispersion and three-dimensional (3D) bending-microchannel was developed to successfully generate droplet swarms with an average diameter of 30 µm. Large specific surface area provided high mass transfer efficiency between phases. While the analogous reaction in a traditional stirred tank required > 270 min to achieve a yield of > 99%, in this biphasic microreaction system, the yield reached 99.6% with a high enantiomeric excess (ee) of > 99% in only 80 min. Efficient synthesis was achieved by reducing the time by 70%.


Asunto(s)
Escherichia coli , Metanol , Biocatálisis , Estereoisomerismo
2.
Food Chem ; 351: 129335, 2021 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-33662910

RESUMEN

The effect of hydrolysis degree of gallotannins (GT, 1 mg/g) on cross-linking of nano-size collagen catalyzed by laccase (12 U/g) was studied, and the antibacterial properties of GT hydrolysates (HGT)-laccase (Lac) collagen films on minced cod were also investigated. The results showed that the tensile strength of HGT-Lac films (87.23-100.77 MPa) was higher than those added HGT alone (85.59-95.58 MPa) under the same hydrolysis degree of GT. Compared to the denaturation temperature (78.05 °C) of pure nano-size collagen film without addition of HGT and laccase, the denaturation temperature of HGT (80.75-86.30 °C) and HGT-Lac (91.97-101.64 °C) films increased greatly, especially for HGT-Lac films. Moreover, both HGT and HGT-Lac films showed some mild antibacterial properties for minced cod during storage at 4 °C for 8 days. Therefore, the combination of HGT and laccase could improve the performance of nano-size collagen film and extend the application of collagen in biodegradable/edible packaging.


Asunto(s)
Biocatálisis , Colágeno/química , Colágeno/metabolismo , Taninos Hidrolizables/química , Lacasa/metabolismo , Películas Comestibles , Temperatura , Resistencia a la Tracción
3.
Nat Commun ; 12(1): 1782, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741963

RESUMEN

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.


Asunto(s)
Macrólidos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biocatálisis/efectos de los fármacos , Bovinos , Microscopía por Crioelectrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Macrólidos/química , Macrólidos/metabolismo , Modelos Moleculares , Estructura Molecular , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/ultraestructura
4.
Nat Commun ; 12(1): 1228, 2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33623032

RESUMEN

Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.


Asunto(s)
Bdellovibrio bacteriovorus/enzimología , Malato Deshidrogenasa/metabolismo , Acetilcoenzima A/metabolismo , Regulación Alostérica , Apoproteínas/química , Sitios de Unión , Biocatálisis , Cinética , Malato Deshidrogenasa/química , Modelos Moleculares , Movimiento (Física) , Dominios Proteicos
5.
Food Chem ; 349: 129127, 2021 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-33561794

RESUMEN

Metal-organic structures (MOFs) have been designed for a wide range of applications due to their high porosity, large surface area, and flexibility. For the first time in this work, the successful immobilization of α-amylase is confirmed by the use of ZIF-8 as easy and good support. The morphology, functional groups, and chemical composition of the support and immobilized α-amylase were tested using different methods such as scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analysis (TGA). The enzymatic activities of the immobilized olibanum-bovine serum albumin@zeolitic imidazolate frameworks nanocomposite (OLB/BSA@ZIF-8)-α-amylase were compared with the free one. The pH and thermal stability of the OLB/BSA@ZIF-8-α-amylase were significantly enhanced compared to the free enzyme. The OLB/BSA@ZIF-8-α-amylase displayed excellent long-term storage stability, which could protect more than 90% of the initial activity for 8 weeks. Besides, the OLB/BSA@ZIF-8-α-amylase had high reusability, which showed a high degree of activity (more than 81%) after 20 cycles. This is the first study that uses OLB/BSA@ZIF-8 nanocomposite as immobilizing support for the immobilization of α-amylase. Improved catalytic efficiency (Vmax/Km) values, reusability, and storage stability of immobilized α-amylase can make it suitable in industrial and biotechnological applications.


Asunto(s)
Enzimas Inmovilizadas/química , Estructuras Metalorgánicas/química , Nanocompuestos/química , Albúmina Sérica Bovina/química , alfa-Amilasas/química , Animales , Biocatálisis , Biotecnología , Bovinos , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Porosidad , Zeolitas/química , alfa-Amilasas/metabolismo
6.
Nat Commun ; 12(1): 1095, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33597523

RESUMEN

Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.


Asunto(s)
Proteínas Bacterianas/metabolismo , Dominio Catalítico , Dioxigenasas/metabolismo , Ingeniería de Proteínas/métodos , Pseudomonas fluorescens/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Biocatálisis , Dioxigenasas/química , Dioxigenasas/genética , Modelos Moleculares , Conformación Proteica , Pseudomonas fluorescens/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
7.
Nat Commun ; 12(1): 1220, 2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-33619271

RESUMEN

Attachment of ubiquitin (Ub) to proteins is one of the most abundant and versatile of all posttranslational modifications and affects outcomes in essentially all physiological processes. RING E3 ligases target E2 Ub-conjugating enzymes to the substrate, resulting in its ubiquitination. However, the mechanism by which a ubiquitin chain is formed on the substrate remains elusive. Here we demonstrate how substrate binding can induce a specific RING topology that enables self-ubiquitination. By analyzing a catalytically trapped structure showing the initiation of TRIM21 RING-anchored ubiquitin chain elongation, and in combination with a kinetic study, we illuminate the chemical mechanism of ubiquitin conjugation. Moreover, biochemical and cellular experiments show that the topology found in the structure can be induced by substrate binding. Our results provide insights into ubiquitin chain formation on a structural, biochemical and cellular level with broad implications for targeted protein degradation.


Asunto(s)
Biocatálisis , Dominios RING Finger , Ubiquitinación , Animales , Femenino , Masculino , Ratones , Modelos Moleculares , Células 3T3 NIH , Proteolisis , Ribonucleoproteínas/química , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina/metabolismo
8.
Nat Commun ; 12(1): 804, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547322

RESUMEN

Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chemical ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also observed. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.


Asunto(s)
Amino Alcoholes/metabolismo , Butileno Glicoles/metabolismo , ADN/genética , Imidazoles/química , Ácidos Nucleicos/síntesis química , ARN/genética , Amino Alcoholes/química , Emparejamiento Base , Biocatálisis , Butileno Glicoles/química , Cationes Bivalentes , ADN/química , ADN/metabolismo , Cartilla de ADN/química , Cartilla de ADN/metabolismo , Manganeso/química , Manganeso/metabolismo , Conformación de Ácido Nucleico , ARN/química , ARN/metabolismo , Soluciones
9.
Nat Commun ; 12(1): 867, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558520

RESUMEN

Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by lovastatin nonaketide synthase (LovB), with the assistance of a separate trans-acting enoyl reductase (LovC). A full DML synthesis comprises 8 polyketide synthetic cycles with about 35 steps. The assembling of the LovB-LovC complex, and the structural basis for the iterative and yet permutative functions of the megasynthase have remained a mystery. Here, we present the cryo-EM structures of the LovB-LovC complex at 3.60 Å and the core LovB at 2.91 Å resolution. The domain organization of LovB is an X-shaped face-to-face dimer containing eight connected domains. The binding of LovC laterally to the malonyl-acetyl transferase domain allows the completion of a L-shaped catalytic chamber consisting of six active domains. This architecture and the structural details of the megasynthase provide the basis for the processing of the intermediates by the individual catalytic domains. The detailed architectural model provides structural insights that may enable the re-engineering of the megasynthase for the generation of new statins.


Asunto(s)
Lovastatina/biosíntesis , Lovastatina/química , Biocatálisis , Modelos Moleculares , Naftalenos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/ultraestructura , Dominios Proteicos , Especificidad por Sustrato
10.
Nat Commun ; 12(1): 842, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558542

RESUMEN

Discovering autocatalytic chemistries that can evolve is a major goal in systems chemistry and a critical step towards understanding the origin of life. Autocatalytic networks have been discovered in various chemistries, but we lack a general understanding of how network topology controls the Darwinian properties of variation, differential reproduction, and heredity, which are mediated by the chemical composition. Using barcoded sequencing and droplet microfluidics, we establish a landscape of thousands of networks of RNAs that catalyze their own formation from fragments, and derive relationships between network topology and chemical composition. We find that strong variations arise from catalytic innovations perturbing weakly connected networks, and that growth increases with global connectivity. These rules imply trade-offs between reproduction and variation, and between compositional persistence and variation along trajectories of network complexification. Overall, connectivity in reaction networks provides a lever to balance variation (to explore chemical states) with reproduction and heredity (persistence being necessary for selection to act), as required for chemical evolution.


Asunto(s)
Biocatálisis , Redes y Vías Metabólicas , ARN/metabolismo
11.
Bioresour Technol ; 329: 124832, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33631450

RESUMEN

This study aimed to develop a biotransformation process for the production of (S)-1-[2-(trifluoromethyl)phenyl]ethanol, a key chiral intermediate of Plk1 inhibitor, and increase its productivity through medium engineering strategy. A fungus isolate Geotrichum silvicola ZJPH1811 was adopted as biocatalyst for 2'-(trifluoromethyl)acetophenone reduction, and gave the best performance with > 99.2% product ee. To improve the yield, choline acetate/cysteine (ChAc/Cys) was introduced as co-solvent in reaction system, which accelerated mass transfer and protected cells from substrate inhibition. Moreover, a synergistic effect of methylated-ß-cyclodextrin (MCD) and ChAc/Cys was found in the bioreduction, with further enhancement in substrate concentration and cell membrane permeability. Compared with buffer system, in the developed ChAc/Cys-MCD-containing system, substrate loading and product yield were increased by 6.7-fold and 2.4-fold respectively. This is the first report on (S)-1-[2-(trifluoromethyl)phenyl]ethanol production with G. silvicola, and provides valuable insight into the synergistic effect of DES and CDs in biocatalysis.


Asunto(s)
Ciclodextrinas , Alcohol Feniletílico , Biocatálisis , Geotrichum , Solventes
12.
Nature ; 590(7846): 504-508, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33536620

RESUMEN

Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)1-3. The FGFR1 gene is the main candidate driver of tumorigenesis within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.


Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Nucleares/metabolismo , Animales , Biocatálisis , Carcinogénesis/genética , Carcinoma de Células Escamosas/genética , Femenino , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Metilación , Ratones , Modelos Moleculares , Mutación , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/deficiencia , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Food Chem ; 347: 129070, 2021 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-33482483

RESUMEN

Interesterification is widely employed as an effective technique to modify oils and fats. This study utilizes palm-based oil (palm olein: palm kernel oil: palm stearin, 5:3:2, w/w/w) as the raw material for the interesterification process performed in a pilot-scale packed bed reactor. Enzymatic interesterification (EIE) was catalyzed by Lipozyme TL IM (813.0 g) at 60℃ with reaction flow rate of 100 mL/min. Chemical interesterification (CIE) was catalyzed using sodium methoxide (0.3 wt%) as catalyst at 105 °C for 30 min. The results showed that the EIE fats had lower solid fat content tendency compared to that of CIE fats. The crystallization onset temperature was higher in EIE fats (23.09℃) compared to that of CIE (19.08℃). The results were consistent with the crystallization kinetics whereby the Avrami K constants of EIE fats were higher than that of CIE fats at various temperatures, indicating rapid crystallization and instant nucleation. Linear growth mechanism was dominant and the crystals formed were smaller in size as observed using polarized light microscope. The interesterified fats exhibited the presence of ß and ß'-crystals. While most of the tocopherol content was retained after EIE (386.18 ug/g), the molecular distillation process reduced the tocopherol concentration (110.01 ug/g) which consequently affected the oxidative stability. The findings in this work contribute to the fundamental understanding on the differences between CIE and EIE fats and provides data to support the preparation of modified fats via EIE that shows great potential as a controllable technique for industrialization.


Asunto(s)
Ácidos Grasos/química , Aceite de Palma/química , Biocatálisis , Cristalización , Esterificación , Cinética , Lipasa/metabolismo , Ácido Oléico/química , Oxidación-Reducción , Reología , Ácidos Esteáricos/química , Termodinámica
14.
J Agric Food Chem ; 69(2): 815-823, 2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33404235

RESUMEN

Thermostability is a key property of industrial enzymes. Endo-polygalacturonases of the glycoside hydrolase family 28 have many practical applications, but only few of their structures have been determined, and the reasons for their stability remain unclear. We identified and characterized the Talaromyces leycettanus JCM12802 endo-polygalacturonase TlPGA, which differs from other GH28 family members because of its high catalytic activity, with an optimum temperature of 70 °C. Distinctive features were revealed by comparison of thermophilic TlPGA and all known structures of fungal endo-polygalacturonases, including a relatively large exposed polar accessible surface area in thermophilic TlPGA. By mutating potentially important residues in thermophilic TlPGA, we identified Thr284 as a critical residue. Mutant T284A was comparable to thermophilic TlPGA in melting temperature but exhibited a significantly lower half-life and half-inactivation temperature, implicating residue Thr284 in the kinetic stability of thermophilic TlPGA. Structure analysis of thermophilic TlPGA and mutant T284A revealed that a carbon-oxygen hydrogen bond between the hydroxyl group of Thr284 and the Cα atom of Gln255, and the stable conformation adopted by Gln255, contribute to its kinetic stability. Our results clarify the mechanism underlying the kinetic stability of GH28 endo-polygalacturonases and may guide the engineering of thermostable enzymes for industrial applications.


Asunto(s)
Proteínas Fúngicas/química , Poligalacturonasa/química , Talaromyces/enzimología , Secuencia de Aminoácidos , Biocatálisis , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Poligalacturonasa/genética , Poligalacturonasa/metabolismo , Conformación Proteica , Especificidad por Sustrato , Talaromyces/química , Talaromyces/genética , Temperatura
15.
Carbohydr Polym ; 256: 117510, 2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33483031

RESUMEN

Cellulose nanofibers (CNF) are renewable and biodegradable nanomaterials with attractive barrier, mechanical and surface properties. In this work, three different recombinant enzymes: an endoglucanase, a xylanase and a lytic polysaccharide monooxygenase, were combined to enhance cellulose fibrillation and to produce CNF from sugarcane bagasse (SCB). Prior to the enzymatic catalysis, SCB was chemically pretreated by sodium chlorite and KOH, while defibrillation was accomplished via sonication. We obtained much longer (µm scale length) and more thermostable (resisting up to 260 °C) CNFs as compared to the CNFs prepared by TEMPO-mediated oxidation. Our results showed that a cooperative action of the set of hydrolytic and oxidative enzymes can be used as a "green" treatment prior to the sonication step to produce nanofibrillated cellulose with advanced properties.


Asunto(s)
Celulasa/química , Celulosa/química , Endo-1,4-beta Xilanasas/química , Oxigenasas de Función Mixta/química , Nanofibras/química , Biocatálisis , Biodegradación Ambiental , Cloruros/química , Óxidos N-Cíclicos/química , Tecnología Química Verde , Humanos , Hidrólisis , Hidróxidos/química , Nanofibras/ultraestructura , Oxidación-Reducción , Polisacáridos/química , Compuestos de Potasio/química , Saccharum/química , Sonicación
16.
Science ; 371(6524): 64-67, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33384372

RESUMEN

During cell division, kinetochores couple chromosomes to spindle microtubules. To protect against chromosome gain or loss, kinetochores lacking microtubule attachment locally catalyze association of the checkpoint proteins Cdc20 and Mad2, which is the key event in the formation of a diffusible checkpoint complex that prevents mitotic exit. We elucidated the mechanism of kinetochore-catalyzed Mad2-Cdc20 assembly with a probe that specifically monitors this assembly reaction at kinetochores in living cells. We found that catalysis occurs through a tripartite mechanism that includes localized delivery of Mad2 and Cdc20 substrates and two phosphorylation-dependent interactions that geometrically constrain their positions and prime Cdc20 for interaction with Mad2. These results reveal how unattached kinetochores create a signal that ensures genome integrity during cell division.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Cdc20/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Animales , Biocatálisis , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Mitosis , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
17.
Science ; 371(6524): 67-71, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33384373

RESUMEN

Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.


Asunto(s)
Proteínas Cdc20/metabolismo , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2/metabolismo , Complejos Multiproteicos/metabolismo , Biocatálisis , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Proteínas Mad2/genética , Mutación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Serina-Treonina Quinasas , Huso Acromático/metabolismo
18.
Chemistry ; 27(17): 5404-5411, 2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33496351

RESUMEN

Indigoids represent natural product-based compounds applicable as organic semiconductors and photoresponsive materials. Yet modified indigo derivatives are difficult to access by chemical synthesis. A biocatalytic approach applying several consecutive selective C-H functionalizations was developed that selectively provides access to various indigoids: Enzymatic halogenation of l-tryptophan followed by indole generation with tryptophanase yields 5-, 6- and 7-bromoindoles. Subsequent hydroxylation using a flavin monooxygenase furnishes dibromoindigo that is derivatized by acylation. This four-step one-pot cascade gives dibromoindigo in good isolated yields. Moreover, the halogen substituent allows for late-stage diversification by cross-coupling directly performed in the crude mixture, thus enabling synthesis of a small set of 6,6'-diarylindigo derivatives. This chemoenzymatic approach provides a modular platform towards novel indigoids with attractive spectral properties.


Asunto(s)
Halogenación , Triptófano , Biocatálisis , Flavinas , Halógenos , Triptófano/metabolismo
19.
Nature ; 590(7847): 624-629, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33461211

RESUMEN

In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.


Asunto(s)
Nucleótidos de Adenina/metabolismo , Sistemas CRISPR-Cas/inmunología , ADN de Cadena Simple/metabolismo , Desoxirribonucleasas/metabolismo , Endorribonucleasas/metabolismo , Oligorribonucleótidos/metabolismo , ARN/metabolismo , Staphylococcus/enzimología , Staphylococcus/inmunología , Nucleótidos de Adenina/inmunología , Adenosina Trifosfato/metabolismo , Bacteriófagos/inmunología , Bacteriófagos/fisiología , Biocatálisis , Dominio Catalítico , Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Endorribonucleasas/química , Endorribonucleasas/genética , Activación Enzimática , Ligandos , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Oligorribonucleótidos/inmunología , Plásmidos/genética , Plásmidos/metabolismo , Multimerización de Proteína , Rotación , Staphylococcus/crecimiento & desarrollo , Staphylococcus/virología , Especificidad por Sustrato
20.
ACS Appl Mater Interfaces ; 13(5): 6168-6179, 2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33499600

RESUMEN

Developing novel immobilization methods to maximize the catalytic performance of enzymes has been a permanent pursuit of scientific researchers. Engineered Escherichia coli biofilms have attracted great concern as surface display platforms for enzyme immobilization. However, current biological conjugation methods, such as the SpyTag/SpyCatcher tagging pair, that immobilize enzymes onto E. coli biofilms seriously hamper enzymatic performance. Through phage display screening of lipase-binding peptides (LBPs) and co-expression of CsgB (nucleation protein of curli nanofibers) and LBP2-modified CsgA (CsgALBP2, major structural subunit of curli nanofibers) proteins, we developed E. coli BL21::ΔCsgA-CsgB-CsgALBP2 (LBP2-functionalized) biofilms as surface display platforms to maximize the catalytic performance of lipase (Lip181). After immobilization onto LBP2-functionalized biofilm materials, Lip181 showed increased thermostability, pH, and storage stability. Surprisingly, the relative activity of immobilized Lip181 increased from 8.43 to 11.33 U/mg through this immobilization strategy. Furthermore, the highest loading of lipase on LBP2-functionalized biofilm materials reached up to 27.90 mg/g of wet biofilm materials, equivalent to 210.49 mg/g of dry biofilm materials, revealing their potential as a surface with high enzyme loading capacity. Additionally, immobilized Lip181 was used to hydrolyze phthalic acid esters, and the hydrolysis rate against dibutyl phthalate was up to 100%. Thus, LBP2-mediated immobilization of lipases was demonstrated to be far more advantageous than the traditional SpyTag/SpyCatcher strategy in maximizing enzymatic performance, thereby providing a better alternative for enzyme immobilization onto E. coli biofilms.


Asunto(s)
Biopelículas , Escherichia coli/metabolismo , Lipasa/metabolismo , Péptidos/metabolismo , Biocatálisis , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Lipasa/química , Estructura Molecular , Tamaño de la Partícula , Péptidos/química , Unión Proteica , Propiedades de Superficie
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