RESUMO
With their diverse biological activities, peptides are promising candidates for therapeutic applications, showing antimicrobial, antitumour and hormonal signalling capabilities. Despite their advantages, therapeutic peptides face challenges such as short half-life, limited oral bioavailability and susceptibility to plasma degradation. The rise of computational tools and artificial intelligence (AI) in peptide research has spurred the development of advanced methodologies and databases that are pivotal in the exploration of these complex macromolecules. This perspective delves into integrating AI in peptide development, encompassing classifier methods, predictive systems and the avant-garde design facilitated by deep-generative models like generative adversarial networks and variational autoencoders. There are still challenges, such as the need for processing optimization and careful validation of predictive models. This work outlines traditional strategies for machine learning model construction and training techniques and proposes a comprehensive AI-assisted peptide design and validation pipeline. The evolving landscape of peptide design using AI is emphasized, showcasing the practicality of these methods in expediting the development and discovery of novel peptides within the context of peptide-based drug discovery.
Assuntos
Inteligência Artificial , Descoberta de Drogas , Peptídeos , Peptídeos/química , Peptídeos/uso terapêutico , Peptídeos/farmacologia , Descoberta de Drogas/métodos , Humanos , Desenho de Fármacos , Aprendizado de Máquina , Biologia Computacional/métodosRESUMO
Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.
Assuntos
Sesquiterpenos , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismoRESUMO
The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6-C7 to C7-C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name "germacrene F". Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1â11 to 1â10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature-dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror-symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.
Assuntos
Sesquiterpenos , Simulação de Acoplamento Molecular , Ciclização , Espectroscopia de Ressonância Magnética , Sesquiterpenos/químicaRESUMO
Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.
Assuntos
Produtos Biológicos , Diterpenos , Saccharomyces cerevisiae/genética , Diterpenos/química , Sistema Enzimático do Citocromo P-450/genéticaRESUMO
Protein engineering through directed evolution and (semi)rational approaches is routinely applied to optimize protein properties for a broad range of applications in industry and academia. The multitude of possible variants, combined with limited screening throughput, hampers efficient protein engineering. Data-driven strategies have emerged as a powerful tool to model the protein fitness landscape that can be explored in silico, significantly accelerating protein engineering campaigns. However, such methods require a certain amount of data, which often cannot be provided, to generate a reliable model of the fitness landscape. Here, we introduce MERGE, a method that combines direct coupling analysis (DCA) and machine learning (ML). MERGE enables data-driven protein engineering when only limited data are available for training, typically ranging from 50 to 500 labeled sequences. Our method demonstrates remarkable performance in predicting a protein's fitness value and rank based on its sequence across diverse proteins and properties. Notably, MERGE outperforms state-of-the-art methods when only small data sets are available for modeling, requiring fewer computational resources, and proving particularly promising for protein engineers who have access to limited amounts of data.
Assuntos
Aprendizado de Máquina , Engenharia de Proteínas , Proteínas , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Probabilidade , Modelos MolecularesRESUMO
The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.
Assuntos
Células T Matadoras Naturais , Vacinas , Adjuvantes Imunológicos/farmacologia , Galactosilceramidas/farmacologia , Galactosilceramidas/químicaRESUMO
Silyl ether protecting groups are important tools in organic synthesis, ensuring selective reactions of hydroxyl functional groups. Enantiospecific formation or cleavage could simultaneously enable the resolution of racemic mixtures and thus significantly increase the efficiency of complex synthetic pathways. Based on reports that lipases, which today are already particularly important tools in chemical synthesis, can catalyze the enantiospecific turnover of trimethylsilanol (TMS)-protected alcohols, the goal of this study was to determine the conditions under which such a catalysis occurs. Through detailed experimental and mechanistic investigation, we demonstrated that although lipases mediate the turnover of TMS-protected alcohols, this occurs independently of the known catalytic triad, as this is unable to stabilize a tetrahedral intermediate. The reaction is essentially non-specific and therefore most likely completely independent of the active site. This rules out lipases as catalysts for the resolution of racemic mixtures of alcohols through protection or deprotection with silyl groups.
Assuntos
Éteres , Lipase , Éteres/química , Lipase/química , Álcoois/química , Éter , CatáliseRESUMO
Using the M13 phage display, a series of 7- and 12-mer peptides which interact with new sulfobetaine hydrogels are identified. Two peptides each from the 7- and 12-mer peptide libraries bind to the new sulfobetaine hydrogels with high affinity compared to the wild-type phage lacking a dedicated hydrogel binding peptide. This is the first report of peptides binding to zwitterionic sulfobetaine hydrogels and the study therefore opens up the pathway toward new phage or peptide/hydrogel hybrids with high application potential.
Assuntos
Hidrogéis , Peptídeos , Hidrogéis/metabolismo , Peptídeos/metabolismo , Biblioteca de Peptídeos , Bacteriófago M13/genética , Bacteriófago M13/metabolismoRESUMO
Three previously undescribed azepino-indole alkaloids, named purpurascenines A-C (1-3), together with the new-to-nature 7-hydroxytryptophan (4) as well as two known compounds, adenosine (5) and riboflavin (6), were isolated from fruiting bodies of Cortinarius purpurascens Fr. (Cortinariaceae). The structures of 1-3 were elucidated based on spectroscopic analyses and ECD calculations. Furthermore, the biosynthesis of purpurascenine A (1) was investigated by in vivo experiments using 13C-labeled sodium pyruvate, alanine, and sodium acetate incubated with fruiting bodies of C. purpurascens. The incorporation of 13C into 1 was analyzed using 1D NMR and HRESIMS methods. With [3-13C]-pyruvate, a dramatic enrichment of 13C was observed, and hence a biosynthetic route via a direct Pictet-Spengler reaction between α-keto acids and 7-hydroxytryptophan (4) is suggested for the biosynthesis of purpurascenines A-C (1-3). Compound 1 exhibits no antiproliferative or cytotoxic effects against human prostate (PC-3), colorectal (HCT-116), and breast (MCF-7) cancer cells. An in silico docking study confirmed the hypothesis that purpurascenine A (1) could bind to the 5-HT2A serotonin receptor's active site. A new functional 5-HT2A receptor activation assay showed no functional agonistic but some antagonistic effects of 1 against the 5-HT-dependent 5-HT2A activation and likely antagonistic effects on putative constitutive activity of the 5-HT2A receptor.
Assuntos
Cortinarius , Serotonina , Masculino , Humanos , Serotonina/metabolismo , Serotonina/farmacologia , Receptor 5-HT2A de Serotonina , Alcaloides Indólicos/farmacologia , Cortinarius/química , Cortinarius/metabolismoRESUMO
Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. KEY POINTS: ⢠An improvement in protein secretion via the use of T1SS ⢠Reaching almost 400 mg/L of soluble lipase into the supernatant ⢠A step forward to making E. coli cells more competitive for applying as a secretion host.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Sistemas de Secreção Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Lipase/genética , Lipase/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
OBJECTIVES: A better understanding of host-microbe interactions as a cross-talk between the gastrointestinal (GI) tract and the gut microbiota can help treat and prevent GI disorders by improving the maintenance of GI homeostasis. The gut microbiota can affect signaling molecules, such as serotonin, which regulates endocrine systems through the GI tract. Moreover, studying the effects of gut microbiota in the small intestine on the human GI tract health is pivotal. METHODS: Male C57BL/6J mice (n = 30, 10 mice per group) were orally gavaged with 200 µL of PBS (control group); mice in group II were orally gavaged with 109 colony-forming units (CFU)/200 µL of viable A. muciniphila, suspended in PBS (A. muciniphila group); and mice in group III were orally gavaged with 10 µg of protein/200 µL of EVs (A. muciniphila-EV group) once daily for four weeks. The gene expression of serotonin system-related genes (Slc6a4, Tph1, Mao, Htr3, Htr4, and Htr7) was examined by quantitative real-time PCR (qPCR) method. RESULTS: Based on the results, A. muciniphila significantly affected the mRNA expression of genes related to the serotonin system (Tph1, Mao, Htr3B, and Htr7) in the duodenum and (Htr3B, Htr4 and Htr7) in the ileum of mice (P < 0.05). Moreover, A. muciniphila-derived EVs affected the expression of major genes related to the serotonin system (Tph1, slc6a4a, Mao, Htr3B, Htr4, and Htr7) in the duodenum and ileum of mice (P < 0.05). CONCLUSIONS: The present findings may pave the way for further investigation of the effects of strain-specific probiotics on the serotonergic system, which is currently in its infancy.
Assuntos
Vesículas Extracelulares , Serotonina , Camundongos , Masculino , Humanos , Animais , Serotonina/metabolismo , Camundongos Endogâmicos C57BL , Verrucomicrobia/fisiologia , Intestino Delgado , Expressão Gênica , Monoaminoxidase/genética , Monoaminoxidase/metabolismoRESUMO
Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.
Assuntos
Agmatina , Rhizobium , Animais , Histamina/metabolismo , Especificidade por Substrato , Rhizobium/metabolismo , Agmatina/análise , Aminas Biogênicas/análise , Qualidade dos Alimentos , Engenharia de ProteínasRESUMO
Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.
Assuntos
Proteínas de Bactérias/química , Chloroflexus/metabolismo , Flavinas/metabolismo , Proteínas Luminescentes/química , Proteínas Mutantes/química , Mutação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chloroflexus/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Simulação de Dinâmica Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fotoquímica , Conformação Proteica , Teoria QuânticaRESUMO
Two terpene cyclases were used as biocatalytic tool, namely, limonene synthase from Cannabis sativa (CLS) and 5-epi-aristolochene synthase (TEAS) from Nicotiana tabacum. They showed significant substrate flexibility towards non-natural prenyl diphosphates to form novel terpenoids, including core oxa- and thia-heterocycles and alkyne-modified terpenoids. We elucidated the structures of five novel monoterpene-analogues and a known sesquiterpene-analogue. These results reflected the terpene synthases' ability and promiscuity to broaden the pool of terpenoids with structurally complex analogues. Docking studies highlight an on-off conversion of the unnatural substrates.
Assuntos
Alquil e Aril Transferases , Perfumes , Terpenos/metabolismo , Difosfatos/química , Odorantes , Alcinos , Alquil e Aril Transferases/metabolismo , BiotransformaçãoRESUMO
Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Proteínas Hemolisinas , Engenharia de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Regiões Terminadoras Genéticas , Sistemas de Secreção Tipo I/metabolismoRESUMO
The combination of diversity generation methods and ultrahigh-throughput screening (uHTS) technologies is key to efficiently explore nature's sequence space and elucidate structure-function relationships of enzymes. Beneficial substitutions often cluster in a few regions and simultaneous amino acid substitutions at multiple positions (e.g., by OmniChange) will likely lead to further improved enzyme variants. An extensive screening effort is required to identify such variants, as the simultaneous randomization of four codons can easily yield over 105 potential enzyme variants. The combination of flow cytometer-based uHTS with cell-free compartmentalization technology using (w/o/w) double emulsions (InVitroFlow), provides analysis capabilities of up to 107 events per hour, thus enabling efficient screening. InVitroFlow is an elegant solution since diversity loss through a transformation of host cells is omitted and emulsion compartments provide a genotype-phenotype linkage through a fluorescence readout. In this study, a multisite saturation mutagenesis and an OmniChange library with four simultaneously saturated positions in the active site of CelA2 cellulase were screened using InVitroFlow. Screening of over 36 million events, yielded a significantly improved cellulase variant CelA2-M3 (H288F/H524Q) with an 8-fold increase in specific activity compared to the parent CelA2-H288F (83.9 U/mg) and a 41-fold increased specific activity (674.5 U/mg) compared to wildtype CelA2 (16.6 U/mg) for the substrate 4-MUC (4-methylumbelliferyl-ß d-cellobioside).
Assuntos
Celulase , Substituição de Aminoácidos , Celulase/genética , Celulase/metabolismo , Códon , Evolução Molecular Direcionada/métodos , Biblioteca Gênica , MutagêneseRESUMO
Cytochrome P450s are heme-containing enzymes capable of the oxidative transformation of a wide range of organic substrates. A protein scaffold that coordinates the heme iron, and the catalytic pocket residues, together, determine the reaction selectivity and regio- and stereo-selectivity of the P450 enzymes. Different substrates also affect the properties of P450s by binding to its catalytic pocket. Modulating the redox potential of the heme by substituting iron-coordinating residues changes the chemical reaction, the type of cofactor requirement, and the stereoselectivity of P450s. Around hundreds of P450s are experimentally characterized, therefore, a mechanistic understanding of the factors affecting their catalysis is increasingly vital in the age of synthetic biology and biotechnology. Engineering P450s can enable them to catalyze a variety of chemical reactions viz. oxygenation, peroxygenation, cyclopropanation, epoxidation, nitration, etc., to synthesize high-value chiral organic molecules with exceptionally high stereo- and regioselectivity and catalytic efficiency. This review will focus on recent studies of the mechanistic understandings of the modulation of heme redox potential in the engineered P450 variants, and the effect of small decoy molecules, dual function small molecules, and substrate mimetics on the type of chemical reaction and the catalytic cycle of the P450 enzymes.
Assuntos
Sistema Enzimático do Citocromo P-450 , Ferro , Especificidade por Substrato , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredução , Heme/metabolismoRESUMO
Understanding the mechanisms of modulators' action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regulated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM17's broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New 'exosite' (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modulators as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibition mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity.
Assuntos
Proteína ADAM17/antagonistas & inibidores , Proteína ADAM17/efeitos dos fármacos , Proteína ADAM17/metabolismo , Proteínas ADAM/metabolismo , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Cinética , Inibidores de Proteases/farmacologia , Especificidade por Substrato/efeitos dos fármacosRESUMO
The CompassR (computer-assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipaseâ A (BSLA), which improves resistance to the organic cosolvent 1,4-dioxane (DOX), were studied to compare two recombination strategies, the two-gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX-resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6-fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0-fold), 30 % (v/v) ethanol (2.1-fold), and 60 % (v/v) methanol (2.4-fold) compared with wild-type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/ß-hydrolase fold.
Assuntos
Lipase/química , Lipase/genética , Recombinação Genética , Solventes/química , Bacillus subtilis/enzimologia , Evolução Molecular Direcionada , Estabilidade Enzimática/genética , Lipase/metabolismo , Simulação de Dinâmica Molecular , Água/químicaRESUMO
Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases.