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Evolution of P450 BM3 is a topic of extensive research, but screening the various substrate/reaction combinations remains a time-consuming process. Indigo production has the potential to serve as a simple high-throughput method for reaction screening, as bacterial colonies expressing indigo (+) variants can be visually identified via their blue phenotype. Indigo (+) single variants, indigo (-) single variants and a combinatorial library, containing mutations that enable the blue phenotype, were screened for their ability to hydroxylate a panel of 12 aromatic compounds using the 4-aminoantipyrine colorimetric assay. Recombination of indigo (+) single variants to create a multiple-variant library is a particularly useful strategy, as all top performing P450 BM3 variants with high hydroxylation activity were either indigo (+) single variants or contained multiple substitutions. Furthermore, active variants, as determined using the 4-AAP assay, were further characterized and several variants were identified that gave more than 90% conversion with 1,3-dichlorobenzene and predominantly formed 2,6-dichlorophenol; other variants showed significant substrate selectivity. This supports the hypothesis that substitution at positions that enable the indigo (+) phenotype, or hotspot residues, is a general mechanism for increasing aromatic hydroxylation activity. Overall, this research demonstrates that indigo (+) single variants, identified via colorimetric colony-based screening, may be recombined to generate a multiply-substituted variant library containing many variants with high aromatic hydroxylation activity. The combination of colony-based screening and other screening assays greatly accelerates enzyme engineering, as readily-identified indigo (+) single variants can be recombined to create a library of active multiple variants without extensive screening of single variants.
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Engineering studies of Candida (Pseudozyma) antarctica lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or short/medium-chain fatty acid esters. We demonstrate that various mechanisms contribute to the diverse selectivity profiles, ranging from reshaping tunnel morphology and tunnel stabilization to obstructing the main substrate-binding tunnel, highlighting the dynamic nature of the substrate-binding region. This work provides important insights into the versatility of this robust lipase toward diverse applications.
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Ésteres , Lipase , Lipase/química , Hidrólise , Domínio Catalítico , Ésteres/química , Ácidos Graxos/metabolismo , Especificidade por SubstratoRESUMO
COVID-19 represents a multi-system infectious disease with broad-spectrum manifestations, including changes in host metabolic processes connected to the disease pathogenesis. Understanding biochemical dysregulation patterns as a consequence of COVID-19 illness promises to be crucial for tracking disease course and clinical outcomes. Surface-enhanced Raman scattering (SERS) has attracted considerable interest in biomedical diagnostics for the sensitive detection of intrinsic profiles of unique fingerprints of serum biomolecules indicative of SARS-CoV-2 infection in a label-free format. Here, we applied label-free SERS and chemometrics for rapid interrogation of temporal metabolic dynamics in longitudinal sera of mildly infected non-hospitalized patients (n = 22), at 4 and 16 weeks post PCR-positive diagnosis, and compared them with negative controls (n = 8). SERS spectral markers revealed distinct metabolic profiles in patient sera that significantly deviated from the healthy metabolic state at the two sampling time intervals. Multivariate and univariate analyses of the spectral data identified abundance dynamics in amino acids, lipids, and protein vibrations as the key spectral features underlying the metabolic differences detected in convalescent samples and perhaps associated with patient recovery progression. A validation study performed using spontaneous Raman spectroscopy yielded spectral data results that corroborated SERS spectral findings and confirmed the detected disease-specific molecular phenotypes in clinical samples. Label-free SERS promises to be a valuable analytical technique for rapid screening of the metabolic phenotype induced by SARS-CoV-2 infection to allow appropriate healthcare intervention.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Proteínas , Análise Espectral Raman/métodos , MetabolomaRESUMO
Enzyme engineering allows to explore sequence diversity in search for new properties. The scientific literature is populated with methods to create enzyme libraries for engineering purposes, however, choosing a suitable method for the creation of mutant libraries can be daunting, in particular for the novices. Here, we address both novices and experts: how can one enter the arena of enzyme library design and what guidelines can advanced users apply to select strategies best suited to their purpose? Section I is dedicated to the novices and presents an overview of established and standard methods for library creation, as well as available commercial solutions. The expert will discover an up-to-date tool to freshen up their repertoire (Section I) and learn of the newest methods that are likely to become a mainstay (Section II). We focus primarily on in vitro methods, presenting the advantages of each method. Our ultimate aim is to offer a selection of methods/strategies that we believe to be most useful to the enzyme engineer, whether a first-timer or a seasoned user.
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Enzimas/genética , Variação Genética , AprendizagemRESUMO
We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG antibodies in clinical samples. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson's coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Vacinas contra COVID-19 , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus , Ressonância de Plasmônio de SuperfícieRESUMO
Sulfahydantoin-based molecules may provide a means to counteract antibiotic resistance, which is on the rise. These molecules may act as inhibitors of ß-lactamase enzymes, which are key in some resistance mechanisms. In this paper, we report on the synthesis of 6 novel sulfahydantoin derivatives by the key reaction of chlorosulfonyl isocyanate to form α-amino acid derived sulfamides, and their cyclization into sulfahydantoins. The synthesis is rapid and provides the target compounds in 8 steps. We investigated their potential as ß-lactamase inhibitors using two common Class A ß-lactamases, TEM-1 and the prevalent extended-spectrum TEM-15. Two compounds, 3 and 6, show substantial inhibition of the ß-lactamases with IC50 values between 130 and 510 µM and inferred Ki values between 32 and 55 µM.
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Desenvolvimento de Medicamentos , Compostos de Enxofre/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Compostos de Enxofre/síntese química , Compostos de Enxofre/química , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/químicaRESUMO
Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a >2,000-fold increased trimethoprim resistance on overexpression in E. coli Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.
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Anti-Infecciosos Urinários/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Integrons/genética , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Trimetoprima/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologiaRESUMO
General approaches for controlled protein modification are increasingly sought-after in the arena of chemical biology. Here, using bioorthogonal reactions, we present combinatorial chemoenzymatic strategies to effectuate protein labeling. A total of three metal-free conjugations were simultaneously or sequentially incorporated in a one-pot format with microbial transglutaminase (MTG) to effectuate protein labeling. MTG offers the particularity of conjugating residues within a protein sequence rather than at its extremities, providing a route to labeling the native protein. The reactions are rapid and circumvent the incompatibility posed by metal catalysts. We identify the tetrazine ligation as most-reactive for this purpose, as demonstrated by the fluorescent labeling of two proteins. The Staudinger ligation and strain-promoted azide-alkyne cycloaddition are alternatives. Owing to the breadth of labels that MTG can use as a substrate, our results demonstrate the versatility of this system, with the researcher being able to combine specific protein substrates with a variety of labels.
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Biocatálise , Química Click , Transglutaminases/metabolismo , Alcinos/química , Azidas/química , Modelos Moleculares , Conformação Proteica , Transglutaminases/químicaRESUMO
Herein we report the discovery of the benzo[a]imidazo[2,1,5-c,d]indolizine motif displaying tunable emission covering most of the visible spectrum. The polycyclic core is obtained from readily available amides via a chemoselective process involving Tf2O-mediated amide cyclodehydration, followed by intramolecular C-H arylation. Additionally, these fluorescent heterocycles are easily functionalized using electrophilic reagents, enabling divergent access to varied substitution. The effects of said substitution on the compounds' photophysical properties were rationalized by density functional theory calculations. For some compounds, emission wavelengths are directly correlated to the substituent's Hammett constants. Easily introduced nonconjugated reactive functional groups allow the labeling of biomolecules without modification of emissive properties. This work provides a straightforward platform for the synthesis of new moderately bright fluorescent dyes remarkable for their chemical stability, predictability, and unusually high excitation-emission differential.
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Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease.
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Erros Inatos do Metabolismo dos Aminoácidos/genética , Anemia Megaloblástica/genética , Pancitopenia/genética , Tetra-Hidrofolato Desidrogenase/deficiência , Tetra-Hidrofolato Desidrogenase/genética , Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Sequência de Aminoácidos , Anemia Megaloblástica/tratamento farmacológico , Anemia Megaloblástica/patologia , Sequência de Bases , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Feminino , Ácido Fólico/líquido cefalorraquidiano , Deficiência de Ácido Fólico/líquido cefalorraquidiano , Humanos , Lactente , Leucovorina/uso terapêutico , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Pancitopenia/tratamento farmacológico , Pancitopenia/patologia , Linhagem , Conformação Proteica , Homologia de Sequência de Aminoácidos , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
The great importance of amide bonds in industrial synthesis has encouraged the search for efficient catalysts of amide bond formation. Microbial transglutaminase (MTG) is heavily utilized in crosslinking proteins in the food and textile industries, where the side chain of a glutamine reacts with the side chain of a lysine, forming a secondary amide bond. Long alkylamines carrying diverse chemical entities can substitute for lysine as acyl-acceptor substrates, to link molecules of interest onto peptides or proteins. Here, we explore short and chemically varied acyl-acceptor substrates, to better understand the nature of nonnatural substrates that are tolerated by MTG, with the aim of diversifying biocatalytic applications of MTG. We show, for the first time, that very short-chain alkyl-based amino acids such as glycine can serve as acceptor substrates. The esterified α-amino acids Thr, Ser, Cys, and Trp--but not Ile--also showed reactivity. Extending the search to nonnatural compounds, a ring near the amine group--particularly if aromatic--was beneficial for reactivity, although ring substituents reduced reactivity. Overall, amines attached to a less hindered carbon increased reactivity. Importantly, very small amines carrying either the electron-rich azide or the alkyne groups required for click chemistry were highly reactive as acyl-acceptor substrates, providing a robust route to minimally modified, "clickable" peptides. These results demonstrate that MTG is tolerant to a variety of chemically varied natural and nonnatural acyl-acceptor substrates, which broadens the scope for modification of Gln-containing peptides and proteins.
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Aminas/metabolismo , Aminoácidos/metabolismo , Streptomyces/enzimologia , Transglutaminases/metabolismo , Especificidade por SubstratoRESUMO
Cytochrome P450 BM3 monooxygenase is the topic of extensive research as many researchers have evolved this enzyme to generate a variety of products. However, the abundance of information on increasingly diversified variants of P450 BM3 that catalyze a broad array of chemistry is not in a format that enables easy extraction and interpretation. We present a database that categorizes variants by their catalyzed reactions and includes details about substrates to provide reaction context. This database of >1500 P450 BM3 variants is downloadable and machine-readable and includes instructions to maximize ease of gathering information. The database allows rapid identification of commonly reported substitutions, aiding researchers who are unfamiliar with the enzyme in identifying starting points for enzyme engineering. For those actively engaged in engineering P450 BM3, the database, along with this review, provides a powerful and user-friendly platform to understand, predict, and identify the attributes of P450 BM3 variants, encouraging the further engineering of this enzyme.
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Background: Measuring the ability of SARS-CoV-2 antibodies to neutralize live viruses remains an effective approach to quantify the level of protection of individuals. We assessed the neutralization activity against the ancestral SARS-CoV-2, Delta, Omicron BA.1, BA.2, BA.2.12.1, BA.4 and BA.5 strains, in 280 vaccinated restaurant/bar, grocery and hardware store workers in Québec, Canada. Methods: Participants were recruited during the emergence of Omicron BA.1 variant. The neutralizing activity of participant sera was assessed by microneutralization assay. Results: Serum neutralizing antibody (NtAb) titers of all participants against the ancestral SARS-CoV-2 strain were comparable with those against Delta variant (ranges of titers 10-2032 and 10-2560, respectively), however, their response was significantly reduced against Omicron BA.1, BA2, BA.2.12.1, BA.4 and BA.5 (10-1016, 10-1016, 10-320, 10-80 and 10-254, respectively). Individuals who received 2 doses of vaccine had significantly reduced NtAb titers against all SARS-CoV-2 strains compared to those infected and then vaccinated (≥1 dose), vaccinated (≥2 doses) and then infected, or those who received 3 doses of vaccine. Participants vaccinated with 2 or 3 doses of vaccine and then infected had the highest NtAb titers against all SARS-CoV-2 strains tested. Conclusion: We assessed for the first time the NtAb response in food and retail workers. We found that vaccination prior to the emergence of Omicron BA.1 was associated with higher neutralizing activity against pre-Omicron variants, suggesting the importance of updating vaccines to increase antibody response against new SARS-CoV-2 variants. Vaccination followed by infection was associated with higher neutralizing activity against all SARS-CoV-2 strains tested.
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Ionic liquid self-assembled monolayers (SAM) were designed and applied for binding streptavidin, promoting affinity biosensing and enzyme activity on gold surfaces of sensors. The synthesis of 1-((+)-biotin)pentanamido)propyl)-3-(12-mercaptododecyl)-imidazolium bromide, a biotinylated ionic liquid (IL-biotin), which self-assembles on gold film, afforded streptavidin sensing with surface plasmon resonance (SPR). The IL-biotin-SAM efficiently formed a full streptavidin monolayer. The synthesis of 1-(carboxymethyl)-3-(mercaptododecyl)-imidazoliumbromide, a carboxylated IL (IL-COOH), was used to immobilize anti-IgG to create an affinity biosensor. The IL-COOH demonstrated efficient detection of IgG in the nanomolar concentration range, similar to the alkylthiols SAM and PEG. In addition, the IL-COOH demonstrated low fouling in crude serum, to a level equivalent to PEG. The IL-COOH was further modified with N,N'-bis (carboxymethyl)-l-lysine hydrate to bind copper ions and then, chelate histidine-tagged biomolecules. Human dihydrofolate reductase (hDHFR) was chelated to the modified IL-COOH. By monitoring enzyme activity in situ on the SPR sensor, it was revealed that the IL-COOH SAM improved the activity of hDHFR by 24% in comparison to classical SAM. Thereby, IL-SAM has been synthesized and successfully applied to three important biosensing schemes, demonstrating the advantages of this new class of monolayers.
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Técnicas Biossensoriais/métodos , Imidazóis/química , Líquidos Iônicos/química , Humanos , Ressonância de Plasmônio de Superfície/métodosRESUMO
Non-specific adsorption of the molecular components of biofluids is ubiquitous in the area of biosensing technologies, severely limiting the use of biosensors in real-world applications. The surface chemistries developed to prevent non-specific adsorption of crude serum are not necessarily suited for sensing in other biosamples. In particular, the diagnostic potential of differential expression of proteins in tissues makes cell lysate attractive for disease diagnostics using solid biopsies. However, crude cell lysate poses a significant challenge for surface chemistries because of a large concentration of highly adherent lipids. Contrary to the non-specific adsorption in crude serum being suppressed by hydrophilic surfaces, the surface plasmon resonance (SPR) analysis of serine-, aspartic-acid-, histidine-, leucine-, and phenylalanine-based peptide monolayers revealed that hydrophobic and positively charged peptides decreased non-specific adsorption when using lysate from HEK 293FT cells. A polyethylene glycol (PEG) monolayer resulted in 2-fold greater fouling than the best peptide [3-MPA-(His)2(Leu)2(Phe)2-OH] under the same conditions. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis of the adsorbate from cell lysate confirmed that lipids are the main source of non-specific adsorption. Importantly, the mass spectrometry (MS) study revealed that both the number of lipids identified and their intensity decreased with decreasing non-specific adsorption. A peptide monolayer thus provides an efficient mean to suppress non-specific adsorption from this human cell lysate.
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Técnicas Biossensoriais , Ressonância de Plasmônio de Superfície , Adsorção , Técnicas Biossensoriais/instrumentação , Células HEK293 , Humanos , Lipídeos/química , Espectrometria de Massas , Peptídeos/análise , Polietilenoglicóis/química , Ressonância de Plasmônio de Superfície/instrumentação , Propriedades de SuperfícieRESUMO
This critical review presents an introduction to biocatalysis for synthetic chemists. Advances in biocatalysis of the past 5 years illustrate the breadth of applications for these powerful and selective catalysts in conducting key reaction steps. Asymmetric synthesis of value-added targets and other reaction types are covered, with an emphasis on pharmaceutical intermediates and bulk chemicals. Resources of interest for the non-initiated are provided, including specialized websites and service providers to facilitate identification of suitable biocatalysts, as well as references to recent volumes and reviews for more detailed biocatalytic procedures. Challenges related to the application of biocatalysts are discussed, including how 'green' a biocatalytic reaction may be, and trends in biocatalyst improvement through enzyme engineering are presented (152 references).