RESUMEN
Owing to their photosynthetic capabilities, there is increasing interest in utilizing cyanobacteria to convert solar energy into biomass. 2-Deoxy-scyllo-inosose (DOI) is a valuable starting material for the benzene-free synthesis of catechol and other benzenoids. DOI synthase (DOIS) is responsible for the formation of DOI from d-glucose-6-phosphate (G6P) in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics such as neomycin and butirosin. DOI fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source is necessary for high-yield DOI production. We constructed DOI-producing cyanobacteria toward carbon-free and sustainable DOI production. A DOIS gene derived from the butirosin producer strain Bacillus circulans (btrC) was introduced and expressed in the cyanobacterium Synechococcus elongatus PCC 7942. We ultimately succeeded in producing 400 mg/L of DOI in S. elongatus without using a carbon source. DOI production by cyanobacteria represents a novel and efficient approach for producing benzenoids from G6P synthesized by photosynthesis.
Asunto(s)
Inositol/análogos & derivados , Synechococcus/química , Benzaldehídos/química , Benceno/química , Benzoquinonas/química , Sulfato de Butirosina/biosíntesis , Catecoles/química , Inositol/biosíntesis , Neomicina/biosíntesis , FotosíntesisRESUMEN
Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354-363 and 371-379 separated by a more flexible segment of residues 364-370. In LPPG micelles a helical conformation was observed for residues 354-377 with greater flexibility in the 366-367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion.
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Membrana Celular/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Dicroismo Circular , Glucolípidos/química , Glucolípidos/metabolismo , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Micelas , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.
Asunto(s)
Isoquinolinas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Receptores de GABA/metabolismo , Animales , Liposomas , Ratones , Transporte de Proteínas , Tecnicas de Microbalanza del Cristal de CuarzoRESUMEN
Membrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins utilise the Sec translocon as the major facilitator of insertion; however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion; this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F0c (subunit c of the F0F1 ATP synthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F0c and (iii) show that incubation of F0c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F0c through Blue Native PAGE and fluorescence quenching reveal a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Membrana Celular/efectos de los fármacos , Diglicéridos/farmacología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/aislamiento & purificación , Modelos Biológicos , Estructura Cuaternaria de Proteína , ATPasas de Translocación de Protón , Solubilidad , Especificidad por Sustrato/efectos de los fármacosRESUMEN
S100B is a calcium sensing protein belonging to the S100 protein family with intracellular and extracellular roles. It is one of the EF hand homodimeric proteins, which is known to interact with various protein targets to regulate varied biological functions. Extracellular S100B has been recently reported to interact with FGF2 in a RAGE-independent manner. However, the recognition mechanism of S100B-FGF2 interaction at the molecular level remains unclear. In this study, the critical residues on S100B-FGF2 interface were mapped by combined information derived from NMR spectroscopy and site directed mutagenesis experiments. Utilizing NMR titration data, we generated the structural models of S100B-FGF2 complex from the computational docking program, HADDOCK which were further proved stable during 15ns unrestrained molecular dynamics (MD) simulations. Isothermal titration calorimetry studies indicated S100B interaction with FGF2 is an entropically favored process implying dominant role of hydrophobic contacts at the protein-protein interface. Residue level information of S100B interaction with FGF2 was useful to understand the varied target recognition ability of S100B and further explained its role in effecting extracellular signaling diversity. Mechanistic insights into the S100B-FGF2 complex interface and cell-based assay studies involving mutants led us to conclude the novel role of S100B in FGF2 mediated FGFR1 receptor inactivation.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Complejos Multiproteicos/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Transducción de Señal/fisiología , Línea Celular Tumoral , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Espectroscopía de Resonancia Magnética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Subunidad beta de la Proteína de Unión al Calcio S100/química , Subunidad beta de la Proteína de Unión al Calcio S100/genéticaRESUMEN
Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/ßig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.
Asunto(s)
Sustitución de Aminoácidos , Distrofias Hereditarias de la Córnea , Proteínas de la Matriz Extracelular/química , Mutación Missense , Proteolisis , Factor de Crecimiento Transformador beta/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Mycobacterium tuberculosis/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Proteína Transportadora de Acilo/química , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Coenzima A/química , Estabilidad de Enzimas , Escherichia coli , Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Magnesio/química , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/aislamiento & purificaciónRESUMEN
ClpB is a molecular chaperone from the AAA+ superfamily of ATPases, which reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB is essential for infectivity and in-host survival of a number of pathogenic microorganisms, but systematic studies on ClpB from pathogens have not been reported yet. We purified and characterized one of the two ClpB isoforms from the malaria parasite Plasmodium falciparum, PfClpB1. PfClpB1 is targeted to the apicoplast, an essential plastid organelle that is a promising anti-malaria drug target. PfClpB1 contains all characteristic AAA+ sequence motifs, but the middle domain of PfClpB1 includes a 52-residue long non-conserved insert. Like in most AAA+ ATPases, ATP induces self-association of PfClpB1 into hexamers. PfClpB1 catalyzes the hydrolysis of ATP and its ATPase activity is activated in the presence of casein and poly-lysine. Similar to Escherichia coli ClpB, PfClpB1 reactivates aggregated firefly luciferase, but the PfClpB1-mediated aggregate reactivation is inhibited in the presence of E. coli DnaK, DnaJ, and GrpE. The lack of effective cooperation between PfClpB1 and the bacterial DnaK system may arise from the Plasmodium-specific sequence of the ClpB middle domain. Our results indicate that the chaperone activity of PfClpB1 may support survival of Plasmodium falciparum by maintaining the folding status and activity of apicoplast proteins.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Malaria Falciparum/parasitología , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Animales , Luciérnagas/enzimología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Luciferasas de Luciérnaga/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/aislamiento & purificación , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Análisis de Secuencia de ProteínaRESUMEN
A unique [Ni-Fe-S] cluster (C-cluster) constitutes the active center of Ni-containing carbon monoxide dehydrogenases (CODHs). His(261), which coordinates one of the Fe atoms with Cys(295), is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys(295), we constructed CODH-II variants. Ala substitution for the Cys(295) substitution resulted in the decrease of Ni content and didn't result in major change of Fe content. In addition, the substitution had no effect on the ability to assemble a full complement of [Fe-S] clusters. This strongly suggests Cys(295) indirectly and His(261) together affect Ni-coordination in the C-cluster.
Asunto(s)
Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/metabolismo , Cisteína/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Familia de Multigenes , Níquel/metabolismo , Peptococcaceae/enzimología , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Filogenia , Homología de Secuencia de Aminoácido , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Sitios de Unión , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Soluciones , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology.
Asunto(s)
Discapacidad Intelectual/genética , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Ratones , Serina Endopeptidasas/metabolismoRESUMEN
In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.
Asunto(s)
Alcanos/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Represoras/metabolismo , Transcripción Genética , Yarrowia/genética , Yarrowia/metabolismo , Análisis Mutacional de ADN , ADN de Hongos/química , ADN de Hongos/genética , Datos de Secuencia Molecular , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositoles/metabolismo , Unión Proteica , Proteínas Represoras/genética , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Staphylokinase (SAK), also known as staphylococcal fibrinolysin, is a protein with a molecular mass of about 15 kDa produced by Staphylococcus aureus. Staphylokinase is synthesized in the late exponential phase, similar to streptokinase. The current study identified and predicted the protein SAK from Staphylococcus aureus. SAK is a fibrinolytic enzyme of the third generation that acts as an indirect activator of plasminogen. The current study cloned and expressed SAK protein isolated from Staphylococcus aureus and used in the form of a grid for enhancement of SAK Catalyst with PCR, disengagement, and change into articulation vector PET24b(+). The recombinant plasmid was changed into E. coli strain BL21 (codon additionally to 440) acceptance with isopropyl ß-D-1-thiogalactopyranoside (IPTG).
Asunto(s)
Escherichia coli , Staphylococcus aureus , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Metaloendopeptidasas , Plasminógeno/metabolismo , Staphylococcus aureus/genéticaRESUMEN
Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named RelpLS20, and two auxiliary DNA-binding factors, named Aux1pLS20 and Aux2pLS20. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1pLS20 and the atomic X-ray structure of the C-terminal domain of Aux2pLS20. We also study the interactions between the auxiliary proteins and the full-length RelpLS20, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1pLS20 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2pLS20 shows that it forms a tetramer and suggests that it is an analog of TraMpF of plasmid F. This is the first evidence of the existence of a TraMpF analog in gram positive conjugative systems, although, unlike other TraMpF analogs, Aux2pLS20 does not interact with the relaxase. Aux1pLS20 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase RelpLS20. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.
RESUMEN
Optimization of the fermentation process for recombinant protein production (RPP) is often resource-intensive. Machine learning (ML) approaches are helpful in minimizing the experimentations and find vast applications in RPP. However, these ML-based tools primarily focus on features with respect to amino-acid-sequence, ruling out the influence of fermentation process conditions. The present study combines the features derived from fermentation process conditions with that from amino acid-sequence to construct an ML-based model that predicts the maximal protein yields and the corresponding fermentation conditions for the expression of target recombinant protein in the Escherichia coli periplasm. Two sets of XGBoost classifiers were employed in the first stage to classify the expression levels of the target protein as high (>50 mg/L), medium (between 0.5 and 50 mg/L), or low (<0.5 mg/L). The second-stage framework consisted of three regression models involving support vector machines and random forest to predict the expression yields corresponding to each expression-level-class. Independent tests showed that the predictor achieved an overall average accuracy of 75% and a Pearson coefficient correlation of 0.91 for the correctly classified instances. Therefore, our model offers a reliable substitution of numerous trial-and-error experiments to identify the optimal fermentation conditions and yield for RPP. It is also implemented as an open-access webserver, PERISCOPE-Opt (http://periscope-opt.erc.monash.edu).
RESUMEN
Human enterokinase light chain (hEKL) specifically cleaves the sequence (Asp)4-Lys↓X (D4K), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEKL production from Escherichia coli is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEKL from E. coli by expressing the hEKL variant C112S fused with maltose-binding protein (MBP) through D4K and molecular chaperons including GroEL/ES. The fusion protein self-cleaved in vivo, thereby removing the MBP in the E. coli cells. Thus, the self-cleaved hEKL variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEKL variant exhibiting an enzymatic activity of 3.1 × 103 U/mL (9.934 × 105 U/mg). The approaches presented here greatly simplify the purification of hEKL from E. coli without requiring refolding processes.
RESUMEN
d-xylose reductase is a member of the aldo-keto reductase family, and is involved in d-xylose and l-arabinose conversion through the Pentose Catabolic Pathway (PCP) in fungi. In this study, we biochemically characterized a newly identified second d-xylose reductase (XyrB) from Aspergillus niger. This NADPH-dependent reductase is able to efficiently convert d-xylose and l-arabinose, and it has the highest affinity for these sugars of all currently known fungal pentose reductases. A combination of biochemical data, transcriptomics and phylogenetic analysis further illustrated the role of XyrB in the PCP. Enzymes: D-xylose reductase (EC 1.1.1.307), L-arabinose reductase (EC 1.1.1.21).
RESUMEN
Cytochrome P450 enzymes are a promising tool for the late-stage diversification of lead drug candidates and can provide an alternative route to structural modifications that are difficult to achieve with synthetic chemistry. In this study, a library of P450BM3 mutants was produced using site-directed mutagenesis and the enzymes screened for metabolism of the opium poppy alkaloid noscapine, a drug with anticancer activity. Of the 18 enzyme mutants screened, 12 showed an ability to metabolise noscapine that was not present in the wild-type enzyme. Five noscapine metabolites were detected by LC-MS/MS, with the major metabolite for all mutants being N-demethylated noscapine. The highest observed regioselectivity for N-demethylation was 88%. Two hydroxylated metabolites, a catechol and two C-C cleavage products were also detected. P450-mediated production of hydroxylated and N-demethylated noscapine structures may be useful for the development of noscapine analogues with improved biological activity. The variation in substrate turnover, coupling efficiency and product distribution between the active mutants was considered alongside in silico docking experiments to gain insight into structural and functional effects of the introduced mutations. Selected mutants were identified as targets for further mutagenesis to improve activity and when coupled with an optimised process may provide a route for the preparative-scale production of noscapine metabolites.
RESUMEN
Often glycosidase assays are based on small-molecule compounds where a glycan of interest is linked to a chromophore allowing for easy detection of cleavage of the glycoside bond. However, such compounds only resemble part of the more complex substrate molecule for enzymes acting on glycoconjugates of glycopeptides or glycoproteins. Nonetheless, the advantage is obvious as enzyme activity is readily recorded and kinetic parameters easily obtained. This is not often the case with glycopeptides or glycoproteins as these may reveal increased complexity in terms of heterogeneity in protein-glycan stoichiometry and restricted enzyme accessibility. However, a kinetic analysis of glycan release from glycopeptides could provide information complementary to that of small-molecule substrates, especially if providing kinetic parameters that are immediately comparable. We have characterized the steady state kinetics of wild type and mutant variants of Bifidobacterium longum endo-α-N-acetylgalactosaminidase, by recording the enzymatic release of Galß(1-3)GalNAc from bovine glycomacropeptide pre-treated with sialidase to remove sialic acid units. Differences between previously reported kinetic constants obtained with synthetic substrates and those obtained in the present work demonstrate an influence of the peptide moiety on the kinetic properties of endo-α-N-acetylgalactosaminidase. The devised assay and data handling method determines the accessible substrate concentration as well as the steady state kinetic parameters, KM and kcat, for glycoconjugates of glycopeptides described by the same units as obtained from using small-molecule substrates and thus allows for a direct comparison.
Asunto(s)
Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Biocatálisis , Polisacáridos/química , alfa-N-Acetilgalactosaminidasa/metabolismo , Bifidobacterium longum/enzimología , Glicopéptidos/química , Glicopéptidos/metabolismo , Cinética , Especificidad por SustratoRESUMEN
Mutations in certain genes of the Ribonuclease (RNASE) superfamily can cause amyotrophic lateral sclerosis (ALS) through altered RNA processing mechanisms. About 30 of these missense mutations in RNASE5/ANG gene have already been reported in ALS patients. In another gene of the ribonuclease superfamily, ribonuclease 4 (RNASE4), missense mutations and single nucleotide polymorphisms have been identified in patients suffering from ALS. However, their plausible molecular mechanisms of association with ALS are not known. Here, we present the molecular mechanisms of RNASE4 polymorphisms with ALS using all-atom molecular dynamics (MD) simulations followed by functional assay experiments. As most ALS causing mutations in RNASE superfamily proteins affect either the ribonucleolytic or nuclear translocation activity, we examined these functional properties of wild-type and known RNASE4 variants, R10W, A98V, E48D and V75I, using MD simulations. Our simulation predicted that these variants would retain nuclear translocation activity and that E48D would exhibit loss of ribonucleolytic activity, which was subsequently validated by ribonucleolytic assay. Our results give a mechanistic insight into the association of RNASE4 polymorphisms with ALS and show that E48D-RNASE4 would probably be deleterious and cause ALS in individuals harbouring this polymorphism.