RESUMEN
Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.
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Amoníaco-Liasas , Liasas , Amoníaco-Liasas/química , Amoníaco , Histidina Amoníaco-Liasa/químicaRESUMEN
Aromatic ammonia lyases (AALs) are important enzymes for biocatalysis as they enable the asymmetric synthesis of chiral l-α-amino acids from the corresponding α,ß-unsaturated precursors. AALs have very similar protein structures and active site pockets but exhibit strict substrate specificity towards tyrosine, phenylalanine, or histidine. Herein, through systematic bioinformatics and structural analysis, we discovered eight new motifs of amino acid residues in AALs. After introducing them - as well as four already known motifs - into different AALs, we learned that altering the substrate specificity by engineering the substrate switch motif in phenylalanine ammonia lyases (PALs), phenylalanine/tyrosine ammonia lyases (PTALs), and tyrosine ammonia lyases (TALs) was only partially successful. However, we discovered that three previously unknown residue combinations introduced a substrate switch from tyrosine to phenylalanine in TAL, which was converted up to 20-fold better compared to the wild-type TAL enzyme.
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Amoníaco-Liasas , Liasas , Liasas/metabolismo , Amoníaco-Liasas/química , Fenilanina Amoníaco-Liasa/química , Aminoácidos/metabolismo , Fenilalanina , Tirosina , Especificidad por SustratoRESUMEN
Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in NasF5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12â self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively.
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Dicetopiperazinas , Triptófano , Triptófano/química , Dicetopiperazinas/química , Especificidad por Sustrato , Simulación de Dinámica Molecular , DimerizaciónRESUMEN
Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. Machine learning provides a promising approach for protein engineering, but activity prediction models for ATAs remain elusive due to the difficulty of obtaining high-quality training data. Thus, we first created variants of the ATA from Ruegeria sp. (3FCR) with improved catalytic activity (up to 2000-fold) as well as reversed stereoselectivity by a structure-dependent rational design and collected a high-quality dataset in this process. Subsequently, we designed a modified one-hot code to describe steric and electronic effects of substrates and residues within ATAs. Finally, we built a gradient boosting regression tree predictor for catalytic activity and stereoselectivity, and applied this for the data-driven design of optimized variants which then showed improved activity (up to 3-fold compared to the best variants previously identified). We also demonstrated that the model can predict the catalytic activity for ATA variants of another origin by retraining with a small set of additional data.
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Ingeniería de Proteínas , Transaminasas , Transaminasas/metabolismo , Especificidad por Sustrato , Aminas/química , BiocatálisisRESUMEN
Covering: up to 2022Pyrroloindoline is a privileged tricyclic indoline motif widely present in many biologically active and medicinally valuable natural products. Thus, understanding the biosynthesis of this molecule is critical for developing convenient synthetic routes, which is highly challenging for its chemical synthesis due to the presence of rich chiral centers in this molecule, especially the fully substituted chiral carbon center at the C3-position of its rigid tricyclic structure. In recent years, progress has been made in elucidating the biosynthetic pathways and enzymatic mechanisms of pyrroloindoline-containing natural products (PiNPs). This article reviews the main advances in the past few decades based on the different substitutions on the C3 position of PiNPs, especially the various key enzymatic mechanisms involved in the biosynthesis of different types of PiNPs.
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Productos Biológicos , Productos Biológicos/química , Vías Biosintéticas , CarbonoRESUMEN
p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l-tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TALclu ) has the highest affinity (Km =0.019â mm) and conversion efficiency (kcat /Km= 1631 s-1 â mm-1 ) towards l-tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03â g/L after 8â hours by TALclu and 2.35â g/L after 24â h by TAL from Rivularia sp. PCC 7116 (TALrpc ) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.
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Amoníaco-Liasas , Escherichia coli , Amoníaco-Liasas/genética , Ácidos Cumáricos , Escherichia coli/metabolismo , Fenilanina Amoníaco-Liasa , Filogenia , Tirosina/metabolismoRESUMEN
Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. P450-mediated biocatalysis offers a practical avenue to access their structural diversity; however, many of these enzymes are insoluble in Escherichia coli and difficult to operate in Streptomyces. Through validation of the functions of two pairs Mycobacterium smegmatis sourced redox partners in vitro, and comparing the efficiency of different biocatalytic systems with tricky P450s in vivo, we herein demonstrated that M. smegmatis is much more efficient, robust, and cleaner in metabolites background than the regularly used E. coli or Streptomyces systems. The M. smegmatis-based system can completely convert 1 g L-1 of cyclodipeptide into HTDKPs within 18 h with minimal background metabolites. On the basis of this efficient system, 12 novel HTDPKs were readily obtained by using two HTDKP-forming P450s (NasbB and NASS1868). Among them, five compounds have neuroprotective properties. Our study significantly expands the bioactive chemical scope of HTDKPs and provides an excellent biocatalysis platform for dealing with problematic enzymes from Actinomycetes.
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Mycobacterium , Streptomyces , Biocatálisis , Dicetopiperazinas , Escherichia coliRESUMEN
Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70-1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.
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Bacterias/metabolismo , Productos Biológicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Dicetopiperazinas/metabolismo , Secuencia de Aminoácidos , Bacterias/genética , Biocatálisis , Productos Biológicos/química , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Dicetopiperazinas/química , Dimerización , Simulación de Dinámica Molecular , Estructura Molecular , Dominios Proteicos , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por Sustrato , Triptófano/químicaRESUMEN
Herein, we report a biocatalytic approach to synthesize plant tetrahydroisoquinoline alkaloids (THIQAs) from dihydroisoquinoline (DHIQ) precursors using imine reductases and N-methyltransferase (NMT). The imine reductase IR45 was engineered to significantly expand its substrate specificity, enabling efficient and stereoselective conversion of 1-phenyl and 1-benzyl 6,7-dimethoxy-DHIQs into the corresponding (S)-tetrahydroisoquinolines (S-THIQs). Coclaurine N-methyltransferase (CNMT) was able to further efficiently convert these (S)-THIQ intermediates into (S)-THIQAs. By assembling IRED, CNMT, and glucose dehydrogenase (GDH) in one reaction, we effectively constituted two artificial biosynthetic pathways in Escherichia coli and successfully applied them to the production of five (S)-THIQAs. This highly efficient (100% yield from DHIQs) and easily tailorable (adding other genes) biosynthetic approach will be useful for producing a variety of plant THIQAs.
RESUMEN
Many natural products contain the hexahydropyrrolo[2, 3-b]indole (HPI) framework. HPI containing chemicals exhibit various biological activities and distinguishable structural arrangement. This structural complexity renders chemical synthesis very challenging. Here, through investigating the biosynthesis of a naturally occurring C3-aryl HPI, naseseazine C (NAS-C), we identify a P450 enzyme (NascB) and reveal that NascB catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon-carbon bonds with both regio- and stereo-specificity. Surprisingly, the limited freedom is allowed in specificity to generate four types of C3-aryl HPI scaffolds, and two of them were not previously observed. By incorporating NascB into an engineered strain of E. coli, we develop a whole-cell biocatalysis system for efficient production of NAS-C and 30 NAS analogs. Interestingly, we find that some of these analogs exhibit potent neuroprotective properties. Thus, our biocatalytic methodology offers an efficient and simple route to generate difficult HPI framework containing chemicals.
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Alcaloides/biosíntesis , Alcaloides/metabolismo , Alcaloides/química , Sistema Enzimático del Citocromo P-450/metabolismo , Dimerización , Escherichia coli/metabolismo , Estructura MolecularRESUMEN
Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report high-resolution X-ray crystal structures of the broadly selective acyltransferase (AT) from the splenocin polyketide synthase (SpnD-AT) in the apo form and in complex with benzylmalonyl and pentynylmalonyl extender unit mimics. These structures revealed the molecular basis for the stereoselectivity and substrate specificity of SpnD-AT, and enabled the engineering of the industrially important Ery-AT6 to broaden its substrate scope to include three new types of extender units.
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Aciltransferasas/química , Sintasas Poliquetidas/química , Aciltransferasas/metabolismo , Estructura Molecular , Sintasas Poliquetidas/metabolismo , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
The preparation of 2'-deoxy-2'-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2'-position. Then, a series of novel 2'-deoxy-2'-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro.
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Antivirales/síntesis química , Nucleósidos/síntesis química , Compuestos de Espiro/síntesis química , Antivirales/farmacología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Hepacivirus/efectos de los fármacos , Humanos , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/farmacología , Virus de la Influenza A/efectos de los fármacos , Concentración 50 Inhibidora , Nucleósidos/farmacología , Compuestos de Espiro/farmacologíaRESUMEN
Among many subtypes of influenza A viruses, influenza A(H1N1) and A(H3N2) subtypes are currently circulating among humans (WHO report 2014-15). Therapeutically, the emergence of viral resistance to currently available drugs (adamantanes and neuraminidase inhibitors) has heightened alarms for developing novel drugs that could address diverse targets in the viral replication cycle in order to improve treatment outcomes. To this regard, the design and synthesis of nucleoside analog inhibitors as potential anti-influenza A agents is a very active field of research nowadays. In this study, we designed and synthesized a series of hitherto unknown 6-methyl-7-substituted-7-deaza purine nucleoside analogs, and evaluated for their biological activities against influenza A virus strains, H1N1 and H3N2. From the viral inhibition assay, we identified some effective compounds, among which, compounds 5x (IC50 = 5.88 µM and 6.95 µM for H1N1 and H3N2, respectively) and 5z (IC50 = 3.95 µM and 3.61 µM for H1N1 and H3N2, respectively) demonstrated potent anti-influenza A activity. On the basis of selectivity index, we conceive that compound 5x may serve as a chemical probe of interest for further lead optimization studies with a general aim of developing novel and effective anti-influenza A virus agents.
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Antivirales/síntesis química , Antivirales/farmacología , Descubrimiento de Drogas , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Antivirales/química , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Oseltamivir/farmacología , Nucleósidos de Purina/química , Ribavirina/farmacología , Zanamivir/farmacologíaRESUMEN
Efficient flow of red blood cells (RBCs) and white blood cells (WBCs) through the microcirculation is necessary for oxygen and nutrient delivery as well as immune cell function. Because blood is a dense particulate suspension, consisting of 40% RBCs by volume, it is difficult to analyze the physical mechanisms by which individual blood cells contribute to the bulk flow properties of blood. Both experimental and computational approaches are hindered by these non-Newtonian properties, and predicting macroscopic blood flow characteristics such as viscosity has historically been an empirical process. In order to examine the effect of the individual cells on macroscopic blood rheology, we developed a lattice Boltzmann model that considers the blood as a suspension of particles in plasma, accounting explicitly for cell-cell and cell-wall interactions. Previous studies have concluded that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells and leukocytes as they enter postcapillary expansions. Similar fluid dynamics may be involved in the initiation of rolling at branch points, a phenomenon linked to atherosclerosis. The lattice Boltzmann approach is used to analyze the interactions of red and white blood cells as they flow through vascular networks digitized from normal and tumor tissue. A major advantage of the lattice-Boltzmann method is the ability to simulate particulate flow dynamically and in any geometry. Using this approach, we can accurately determine RBC-WBC forces, particle trajectories, the pressure changes in each segment that accompany cellular traffic in the network, and the forces felt by the vessel wall at any location. In this technique, intravital imaging using vascular contrast agents produces the network outline that is fed to the lattice-Boltzmann model. This powerful and flexible model can be used to predict blood flow properties in any vessel geometry and with any blood composition.
RESUMEN
Measurements of thermopower S(a)(T) along the highly conducting a axis and specific heat of the Bechgaard salts (TMTSF)(2)ClO(4) for various cooling rates through the anion ordering temperature T(a) = 24 K were carried out. Sign reversal in S(a)(T) is found below T(a) and it decreases with increasing cooling rate, which is attributed to the change of a narrow band filling level as the temperature and the cooling rates change. The crossover from 2D to 3D in S(a)(T) is observed around 15 K. The onset temperature of anion ordering in S(a)(T) decreases from 29.8 to 24.2 K as the cooling rate increases. Meanwhile, the electronic specific heat coefficient γ has a pronounced change within this temperature region, giving strong evidence for a narrow band contribution. The difference in the specific heat between the quenched and relaxed states follows a T-cubic law from 5 to 24 K, implying a lattice distortion by the ordered anion only. The entropy estimated from the specific heat peak between 28 and 15 K is Rln (4/3) lower than the value Rln2, consistent with the thermopower result that some anions have been ordered far above T(a) for the relaxed state.
RESUMEN
Vessel leakiness is a hallmark of inflammation and cancer. In inflammation, plasma extravasation and leukocyte adhesion occur in a coordinated manner to enable the immune response, but also to maintain tissue perfusion. In tumors, similar mechanisms operate, but they are not well regulated. Therefore, blood perfusion in tumors is non-uniform, and delivery of blood-borne therapeutics is difficult. In order to analyze the interplay among plasma leakage, blood viscosity, and vessel geometry, we developed a mathematical model that explicitly includes blood cells, vessel branching, and focal leakage. The results show that local hemoconcentration due to plasma leakage can greatly increase the flow resistance in individual vascular segments, diverting flow to other regions. Similarly, leukocyte rolling can increase flow resistance by partially blocking flow. Vessel dilation can counter these effects, and likely occurs in inflammation to maintain blood flow. These results suggest that potential strategies for improving perfusion through tumor networks include (i) eliminating non-uniform plasma leakage, (ii) inhibiting leukocyte interactions, and (iii) preventing RBC aggregation in sluggish vessels. Normalization of tumor vessels by anti-angiogenic therapy may improve tumor perfusion via the first two mechanisms.
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Velocidad del Flujo Sanguíneo , Volumen Sanguíneo , Modelos Cardiovasculares , Neoplasias/irrigación sanguínea , Vasculitis/fisiopatología , Animales , Presión Sanguínea , Viscosidad Sanguínea , Simulación por Computador , Hematócrito , HumanosRESUMEN
Historically, predicting macroscopic blood flow characteristics such as viscosity has been an empirical process due to the difficulty in rigorously including the particulate nature of blood in a mathematical representation of blood rheology. Using a two-dimensional lattice Boltzmann approach, we have simulated the flow of red blood cells in a blood vessel to estimate flow resistance at various hematocrits and vessel diameters. By including white blood cells (WBCs) in the flow, we also calculate the increase in resistance due to white cell rolling and adhesion. The model considers the blood as a suspension of particles in plasma, accounting for cell-cell and cell-wall interactions to predict macroscopic blood rheology. The model is able to reproduce the Fahraeus-Lindqvist effect, i.e., the increase in relative apparent viscosity as tube size increases, and the Fahraeus effect, i.e., tube hematocrit is lower than discharge hematocrit. In addition, the model allows direct assessment of the effect of WBCs on blood flow in the microvasculature, reproducing the dramatic increases in flow resistance as WBCs enter short capillary segments. This powerful and flexible model can be used to predict blood flow properties in any vessel geometry and with any blood composition.
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Velocidad del Flujo Sanguíneo/fisiología , Fenómenos Fisiológicos Sanguíneos , Eritrocitos/citología , Eritrocitos/fisiología , Hemorreología/métodos , Leucocitos/citología , Leucocitos/fisiología , Modelos Cardiovasculares , Presión Sanguínea/fisiología , Vasos Sanguíneos/citología , Vasos Sanguíneos/fisiología , Viscosidad Sanguínea , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Simulación por Computador , Hematócrito , Microfluídica/métodos , Tamaño de la PartículaRESUMEN
A three-dimensional compressible lattice Boltzmann model is formulated on a cubic lattice. A very large particle-velocity set is incorporated in order to enable a greater variation in the mean velocity. Meanwhile, the support set of the equilibrium distribution has only six directions. Therefore, this model can efficiently handle flows over a wide range of Mach numbers and capture shock waves. Due to the simple form of the equilibrium distribution, the fourth-order velocity tensors are not involved in the formulation. Unlike the standard lattice Boltzmann model, no special treatment is required for the homogeneity of fourth-order velocity tensors on square lattices. The Navier-Stokes equations were recovered, using the Chapman-Enskog method from the Bhatnagar-Gross-Krook (BGK) lattice Boltzmann equation. The second-order discretization error of the fluctuation velocity in the macroscopic conservation equation was eliminated by means of a modified collision invariant. The model is suitable for both viscous and inviscid compressible flows with or without shocks. Since the present scheme deals only with the equilibrium distribution that depends only on fluid density, velocity, and internal energy, boundary conditions on curved wall are easily implemented by an extrapolation of macroscopic variables. To verify the scheme for inviscid flows, we have successfully simulated a three-dimensional shock-wave propagation in a box and a normal shock of Mach number 10 over a wedge. As an application to viscous flows, we have simulated a flat plate boundary layer flow, flow over a cylinder, and a transonic flow over a NACA0012 airfoil cascade.
RESUMEN
Leukocyte rolling on the vascular endothelium requires initial contact between leukocytes circulating in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms also play a role. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells (RBCs) and leukocytes as they enter postcapillary expansions, but the details of the fluid dynamics have not been elucidated. We have analyzed the interactions of red and white blood cells as they flow from a capillary into a postcapillary venule using a lattice Boltzmann approach. This technique provides the complete solution of the flow field and quantification of the particle-particle forces in a relevant geometry. Our results show that capillary-postcapillary venule diameter ratio, RBC configuration, and RBC shape are critical determinants of the initiation of cell rolling in postcapillary venules. The model predicts that an optimal configuration of the trailing red blood cells is required to drive the white blood cell to the wall.