RESUMO
Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.
Assuntos
Evolução Molecular Direcionada , Engenharia de Proteínas , Evolução Molecular Direcionada/métodos , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Enzimas/genética , Enzimas/química , Enzimas/metabolismoRESUMO
Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.
Assuntos
Engenharia de Proteínas , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Estabilidade Enzimática , BurkholderialesRESUMO
Bacterial ß-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as ß-cellulose, ß-glucans and ß-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed 4 C1 conformation to a distorted 1 S3 /1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OHâ â â nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.
Assuntos
Glicosídeo Hidrolases , Simulação de Dinâmica Molecular , Glicosídeo Hidrolases/metabolismo , Ligação de Hidrogênio , Conformação Proteica , Açúcares , Especificidade por Substrato , Cristalografia por Raios X , CatáliseRESUMO
The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.
Assuntos
Burkholderiales , Hidrolases , Hidrolases/química , Burkholderiales/genética , Burkholderiales/metabolismo , Domínio Catalítico , Mutação , Polietilenotereftalatos/metabolismoRESUMO
Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.
Assuntos
Acetilcoenzima A/química , Acetiltransferases/ultraestrutura , Proteínas Recombinantes/ultraestrutura , Streptomyces/ultraestrutura , Acetilcoenzima A/genética , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Animais , Domínio Catalítico/genética , Linhagem Celular , Cristalografia por Raios X , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mutação/genética , Puromicina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Streptomyces/enzimologiaRESUMO
Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel widely used in optogenetics. Photoactivation triggers a trans-to-cis isomerization of a covalently bound retinal. Ensuing conformational changes open a cation-selective channel. We explore the structural dynamics in the early photocycle leading to channel opening by classical (MM) and quantum mechanical (QM) molecular simulations. With QM/MM simulations, we generated a protein-adapted force field for the retinal chromophore, which we validated against absorption spectra. In a 4-µs MM simulation of a dark-adapted ChR2 dimer, water entered the vestibules of the closed channel. Retinal all-trans to 13-cis isomerization, simulated with metadynamics, triggered a major restructuring of the charge cluster forming the channel gate. On a microsecond time scale, water penetrated the gate to form a membrane-spanning preopen pore between helices H1, H2, H3, and H7. This influx of water into an ion-impermeable preopen pore is consistent with time-resolved infrared spectroscopy and electrophysiology experiments. In the retinal 13-cis state, D253 emerged as the proton acceptor of the Schiff base. Upon proton transfer from the Schiff base to D253, modeled by QM/MM simulations, we obtained an early-M/P2390-like intermediate. Rapid rotation of the unprotonated Schiff base toward the cytosolic side effectively prevents its reprotonation from the extracellular side. From MM and QM simulations, we gained detailed insight into the mechanism of ChR2 photoactivation and early events in pore formation. By rearranging the network of charges and hydrogen bonds forming the gate, water emerges as a key player in light-driven ChR2 channel opening.
Assuntos
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Chlamydomonas reinhardtii/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Água/metabolismo , Diterpenos , Ligação de Hidrogênio , Isomerismo , Luz , Modelos Moleculares , Conformação Proteica , Água/químicaRESUMO
Inorganic surfaces and nanoparticles can accelerate or inhibit the fibrillation process of proteins and peptides, including the biomedically relevant amyloid ß peptide. However, the microscopic mechanisms that determine such an effect are still poorly understood. By means of large-scale, state-of-the-art enhanced sampling molecular dynamics simulations, here we identify an interaction mechanism between the segments 16-22 of the amyloid ß peptide, known to be fibrillogenic by itself, and the Au(111) surface in water that leads to the suppression of fiber-like conformations from the peptide conformational ensemble. Moreover, thanks to advanced simulation analysis techniques, we characterize the conformational selection vs. induced fit nature of the gold effect. Our results disclose an inhibition mechanism that is rooted in the details of the microscopic peptide-surface interaction rather than in general phenomena such as peptide sequestration from the solution.
Assuntos
Peptídeos beta-Amiloides/química , Ouro/química , Nanopartículas Metálicas/química , Fragmentos de Peptídeos/química , Adsorção , Sequência de Aminoácidos , Simulação de Dinâmica Molecular , Nanotecnologia , Conformação Proteica , Propriedades de SuperfícieRESUMO
The catalytic mechanism of retaining glycosyltransferases (ret-GTs) remains a controversial issue in glycobiology. By analogy to the well-established mechanism of retaining glycosidases, it was first suggested that ret-GTs follow a double-displacement mechanism. However, only family 6 GTs exhibit a putative nucleophile protein residue properly located in the active site to participate in catalysis, prompting some authors to suggest an unusual single-displacement mechanism [named as front-face or SNi (substitution nucleophilic internal)-like]. This mechanism has now received strong support, from both experiment and theory, for several GT families except family 6, for which a double-displacement reaction is predicted. In the last few years, we have uncovered the molecular mechanisms of several retaining GTs by means of quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, which we overview in the present work.
Assuntos
Glicosiltransferases/metabolismo , Animais , Glicosídeos/química , Glicosídeos/metabolismo , Glicosilação , Glicosiltransferases/química , Humanos , Modelos Moleculares , Teoria QuânticaRESUMO
New, automated forms of data analysis are required to understand the high-dimensional trajectories that are obtained from molecular dynamics simulations on proteins. Dimensionality reduction algorithms are particularly appealing in this regard as they allow one to construct unbiased, low-dimensional representations of the trajectory using only the information encoded in the trajectory. The downside of this approach is that a different set of coordinates are required for each different chemical system under study precisely because the coordinates are constructed using information from the trajectory. In this paper, we show how one can resolve this problem by using the sketch-map algorithm that we recently proposed to construct a low-dimensional representation of the structures contained in the protein data bank. We show that the resulting coordinates are as useful for analyzing trajectory data as coordinates constructed using landmark configurations taken from the trajectory and that these coordinates can thus be used for understanding protein folding across a range of systems.
Assuntos
Proteínas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bases de Dados de Proteínas , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismoRESUMO
This work examines the conformational ensemble involved in ß-hairpin folding by means of advanced molecular dynamics simulations and dimensionality reduction. A fully atomistic description of the protein and the surrounding solvent molecules is used, and this complex energy landscape is sampled by means of parallel tempering metadynamics simulations. The ensemble of configurations explored is analyzed using the recently proposed sketch-map algorithm. Further simulations allow us to probe how mutations affect the structures adopted by this protein. We find that many of the configurations adopted by a mutant are the same as those adopted by the wild-type protein. Furthermore, certain mutations destabilize secondary-structure-containing configurations by preventing the formation of hydrogen bonds or by promoting the formation of new intramolecular contacts. Our analysis demonstrates that machine-learning techniques can be used to study the energy landscapes of complex molecules and that the visualizations that are generated in this way provide a natural basis for examining how the stabilities of particular configurations of the molecule are affected by factors such as temperature or structural mutations.
Assuntos
Simulação de Dinâmica Molecular , Desdobramento de Proteína , Proteínas/química , Algoritmos , Ligação de Hidrogênio , Estrutura Secundária de ProteínaRESUMO
The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve the ultrafast structural changes in the carbonmonoxy myoglobin complex upon photolysis of the Fe-CO bond. Structural changes appear throughout the protein within 500 femtoseconds, with the C, F, and H helices moving away from the heme cofactor and the E and A helices moving toward it. These collective movements are predicted by hybrid quantum mechanics/molecular mechanics simulations. Together with the observed oscillations of residues contacting the heme, our calculations support the prediction that an immediate collective response of the protein occurs upon ligand dissociation, as a result of heme vibrational modes coupling to global modes of the protein.
Assuntos
Mioglobina/química , Animais , Monóxido de Carbono/química , Cristalografia por Raios X , Heme/química , Cavalos , Ferro/química , Ligantes , Simulação de Dinâmica Molecular , Movimento (Física) , Fotólise , Estrutura Secundária de ProteínaRESUMO
Carbohydrate-active enzymes such as glycoside hydrolases (GHs) and glycosyltransferases (GTs) are of growing importance as drug targets. The development of efficient competitive inhibitors and chaperones to treat diseases related to these enzymes requires a detailed knowledge of their mechanisms of action. In recent years, sophisticated first-principles modeling approaches have significantly advanced in our understanding of the catalytic mechanisms of GHs and GTs, not only the molecular details of chemical reactions but also the significant implications that just the conformational dynamics of a sugar ring can have on these mechanisms. Here we provide an overview of the progress that has been made in the past decade, combining molecular dynamics simulations with density functional theory to solve these sweet mysteries of nature.
Assuntos
Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/metabolismo , Animais , Configuração de Carboidratos , Metabolismo dos Carboidratos , Carboidratos/química , Glicosídeo Hidrolases/química , Glicosiltransferases/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Teoria Quântica , TermodinâmicaRESUMO
Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. ß-xylanases are responsible for the hydrolysis of glycosidic bonds in ß-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in ß-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of ß-xylose to predict the most likely catalytic itineraries followed by ß-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer-Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2SO â [2,5B]Ç â 5S1 and 1S3 â [4H3]Ç â 4C1, which clearly appear in low energy regions of the FEL. Consistently, 2SO and 1S3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the OE â [OS2]Ç â B2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed OE conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases.
RESUMO
Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog-fold acyl-CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzymeâ A moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. A single-displacement acid-base-like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state. They also explain the roles of other conserved active-site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone.
Assuntos
Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Acil Coenzima A/metabolismo , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , PrótonsRESUMO
Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe(3+)-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe(3+)-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe(3+)-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe(3+)-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.
Assuntos
Biliverdina/química , Corynebacterium diphtheriae/química , Cristalografia por Raios X , Heme Oxigenase (Desciclizante)/química , Sítios de Ligação , Corynebacterium diphtheriae/patogenicidade , Difteria/enzimologia , Difteria/microbiologia , Difteria/patologia , Heme/química , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Ligação de Hidrogênio , Ferro/química , Ferro/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Água/químicaRESUMO
Elusive glycosyl-enzyme adduct: Using classical MD simulations and QM/MM metadynamics, the long-time sought glycosyl-enzyme covalent intermediate of a retaining glycosyltransferase, with a putative nucleophile residue in the active site, has been trapped (MD=molecular dynamics; QM/MM=quantum mechanics/molecular mechanics).
Assuntos
Enzimas/química , Glicosiltransferases/química , Teoria Quântica , Simulação por Computador , Enzimas/metabolismo , Glicosiltransferases/metabolismo , Modelos MolecularesRESUMO
Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic resolution structures along the reaction coordinate of an inverting α-mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α-D-mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.
Assuntos
Caulobacter/enzimologia , Teoria Quântica , alfa-Manosidase/química , alfa-Manosidase/metabolismo , Biocatálise , Conformação ProteicaRESUMO
Retaining glycoside hydrolases (GHs), key enzymes in the metabolism of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymatic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-ß-glucanase, one of the most active GHs, belonging to family 16. We show that, in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a (1,4)B/(1)S(3) conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymatic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction ((1,4)B/(1)S(3) -(4)E/(4)H(3) - (4)C(1)) and shows that the transition state is not the point of maximum charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme.
Assuntos
Glicosídeo Hidrolases/metabolismo , Teoria Quântica , Catálise , Glicosídeo Hidrolases/química , Glicosilação , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Especificidade por SubstratoRESUMO
Human AKR (aldo-keto reductase) 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 µM). The kcat values were also low (0.18-0.6 min-1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose kcat was remarkably higher (13 min-1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.