RESUMO
The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.
Assuntos
Alcenos , Ácidos Graxos , Ácidos Graxos/metabolismo , Alcenos/química , Descarboxilação , Sistema Enzimático do Citocromo P-450/metabolismo , OxirreduçãoRESUMO
Cellulose possesses considerable potential for a wide range of sustainable applications. Nanocellulose-based material properties are primarily dependent on the structural surface characteristics of its crystalline planes. Experimental measurements of the affinity of crystalline nanocellulose surfaces with water are scarce and challenging to obtain. Therefore, the relative hydrophilicity of different cellulose allomorphs crystalline planes is often inferred from qualitative assessments of their surface and the exposition of polar groups to the solvent. This work investigates the relative hydrophilicity of cellulose surfaces using molecular dynamics simulations. The behavior of a water droplet laid on different crystal planes was used to determine their relative hydrophilicity. The water molecules fully spread onto highly hydrophilic surfaces. However, a water droplet placed on less hydrophilic surfaces equilibrates as an oblate spheroidal cap allowing the measurement of a contact angle. The results indicate that the Iα (010), Iα (11Ì 0), Iß (010), and Iß (110) faces, as well as the faces of human-made celluloses II and III_I (100), (11Ì 0), (010), and (110) are all highly hydrophilic. They all have a contact angle value inferior to 11°. Not unexpectedly, the Iα (001) and Iß (100) surfaces are less hydrophilic with contact angles of 48 and 34°, respectively. However, the Iß (11Ì 0) plane, often referred to as a hydrophilic surface, forms a contact angle of about 32°. The results are rationalized in terms of structure, exposure of hydroxyl groups to the solvent, and degree of cellulose-cellulose versus cellulose-water hydrogen bonds on each face. The simulations also show that the surface oxidation degree tunes the surface hydrophilicity in a nonlinear manner due to cooperative effects involving water-cellulose interactions. Our study helps us to understand how the degree of hydrophilicity of cellulose emerges from specific structural features of each crystalline surface.
Assuntos
Celulose , Simulação de Dinâmica Molecular , Cristalização , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e HidrofílicasRESUMO
Glycoside hydrolases (GH) cleave carbohydrate glycosidic bonds and play pivotal roles in living organisms and in many industrial processes. Unlike acid-catalyzed hydrolysis of carbohydrates in solution, which can occur either via cyclic or acyclic oxocarbenium-like transition states, it is widely accepted that GH-catalyzed hydrolysis proceeds via a general acid mechanism involving a cyclic oxocarbenium-like transition state with protonation of the glycosidic oxygen. The GH45 subfamily C inverting endoglucanase from Phanerochaete chrysosporium (PcCel45A) defies the classical inverting mechanism as its crystal structure conspicuously lacks a general Asp or Glu base residue. Instead, PcCel45A has an Asn residue, a notoriously weak base in solution, as one of its catalytic residues at position 92. Moreover, unlike other inverting GHs, the relative position of the catalytic residues in PcCel45A impairs the proton abstraction from the nucleophilic water that attacks the anomeric carbon, a key step in the classical mechanism. Here, we investigate the viability of an endocyclic mechanism for PcCel45A using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, with the QM region treated with the self-consistent-charge density-functional tight-binding level of theory. In this mechanism, an acyclic oxocarbenium-like transition state is stabilized leading to the opening of the glucopyranose ring and formation of an unstable acyclic hemiacetal that can be readily decomposed into hydrolysis product. In silico characterization of the Michaelis complex shows that PcCel45A significantly restrains the sugar ring to the 4C1 chair conformation at the -1 subsite of the substrate binding cleft, in contrast to the classical exocyclic mechanism in which ring puckering is critical. We also show that PcCel45A provides an environment where the catalytic Asn92 residue in its standard amide form participates in a cooperative hydrogen bond network resulting in its increased nucleophilicity due to an increased negative charge on the oxygen atom. Our results for PcCel45A suggest that carbohydrate hydrolysis catalyzed by GHs may take an alternative route from the classical mechanism.
Assuntos
Celulase , Celulase/metabolismo , Celulose , Hidrólise , Simulação de Dinâmica Molecular , Teoria QuânticaRESUMO
Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/ß-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.
Assuntos
Proteínas de Bactérias/química , Burkholderiales/enzimologia , Esterases/química , Polietilenotereftalatos/química , Proteínas de Bactérias/genética , Burkholderiales/genética , Cristalografia por Raios X , Esterases/genética , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Cellobiohydrolases (CBHs) are key enzymes for the saccharification of cellulose and play major roles in industrial settings for biofuel production. The catalytic core domain of these enzymes exhibits a long and narrow binding tunnel capable of binding glucan chains from crystalline cellulose and processively hydrolyze them. The binding cleft is topped by a set of loops, which are believed to play key roles in substrate binding and cleavage processivity. Here, we present an analysis of the loop motions of the Trichoderma reesei Cel7A catalytic core domain (TrCel7A) using conventional and accelerated molecular dynamics simulations. We observe that the loops exhibit highly coupled fluctuations and cannot move independently of each other. In the absence of a substrate, the characteristic large amplitude dynamics of TrCel7A consists of breathing motions, where the loops undergo open-and-close fluctuations. Upon substrate binding, the open-close fluctuations of the loops are quenched and one of the loops moves parallel to the binding site, possibly to allow processive motion along the glucan chain. Using microsecond accelerated molecular dynamics, we observe large-scale fluctuations of the loops (up to 37 Å) and the entire exposure of the TrCel7A binding site in the absence of the substrate, resembling an endoglucanase. These results suggest that the initial CBH-substrate contact and substrate recognition by the enzyme are similar to that of endoglucanases and, once bound to the substrate, the loops remain closed for proper enzymatic activity.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Proteínas Fúngicas/química , Trichoderma/enzimologia , Sítios de Ligação , Catálise , Domínio Catalítico , Celulose/química , Celulose 1,4-beta-Celobiosidase/metabolismo , Proteínas Fúngicas/metabolismo , Hidrólise , Cinética , Simulação de Dinâmica Molecular , Movimento (Física) , Ligação ProteicaRESUMO
The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls.
Assuntos
Parede Celular/metabolismo , Celulose/metabolismo , Cycadopsida/metabolismo , Magnoliopsida/metabolismo , Xilanos/metabolismo , Acetilação , Evolução Biológica , Parede Celular/química , Celulose/química , Simulação por Computador , Cycadopsida/química , Magnoliopsida/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Xilanos/químicaRESUMO
Lignocellulosic biomass is mainly constituted by cellulose, hemicellulose, and lignin and represents an important resource for the sustainable production of biofuels and green chemistry materials. Xylans, a common hemicellulose, interact with cellulose and often exhibit various side chain substitutions including acetate, (4-O-methyl) glucuronic acid, and arabinose. Recent studies have shown that the distribution of xylan substitutions is not random, but follows patterns that are dependent on the plant taxonomic family and cell wall type. Here, we use molecular dynamics simulations to investigate the role of substitutions on xylan interactions with the hydrophilic cellulose face, using the recently discovered xylan decoration pattern of the conifer gymnosperms as a model. The results show that α-1,2-linked substitutions stabilize the binding of single xylan chains independently of the nature of the substitution and that Ca2+ ions can mediate cross-links between glucuronic acid substitutions of two neighboring xylan chains, thus stabilizing binding. At high temperature, xylans move from the hydrophilic to the hydrophobic cellulose surface and are also stabilized by Ca2+ cross-links. Our results help to explain the role of substitutions on xylan-cellulose interactions, and improve our understanding of the plant cell wall architecture and the fundamentals of biomass pretreatments.
Assuntos
Acetatos/química , Arabinose/química , Celulose/química , Reagentes de Ligações Cruzadas/química , Ácido Glucurônico/química , Xilanos/química , Biomassa , Cálcio/química , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Microfibrilas/química , Simulação de Dinâmica Molecular , Propriedades de SuperfícieRESUMO
Cellulose, the most abundant biopolymer on Earth, represents a resource for sustainable production of biofuels. Thermochemical treatments make lignocellulosic biomaterials more amenable to depolymerization by exposing cellulose microfibrils to enzymatic or chemical attacks. In such treatments, the solvent plays fundamental roles in biomass modification, but the molecular events underlying these changes are still poorly understood. Here, the 3D-RISM-KH molecular theory of solvation has been employed to analyze the role of water in cellulose aggregation under different thermodynamic conditions. The results show that, under ambient conditions, highly structured hydration shells around cellulose create repulsive forces that protect cellulose microfibrils from aggregating. Under hydrothermal pretreatment conditions, however, the hydration shells lose structure, and cellulose aggregation is favored. These effects are largely due to a decrease in cellulose-water interactions relative to those at ambient conditions, so that cellulose-cellulose attractive interactions become prevalent. Our results provide an explanation to the observed increase in the lateral size of cellulose crystallites when biomass is subject to pretreatments and deepen the current understanding of the mechanisms of biomass modification.
Assuntos
Biomassa , Celulose/química , Solventes/química , Água/química , Temperatura Alta , TermodinâmicaRESUMO
Expansins are disruptive proteins that loosen growing plant cell walls and can enhance the enzymatic hydrolysis of cellulose. The canonical expansin structure consists of one domain responsible for substrate binding (D2) and another domain (D1) of unknown function, but essential for activity. Although the effects of expansins on cell walls and cellulose fibrils are known, the molecular mechanism underlying their biophysical function is poorly understood. Here, we use molecular dynamics simulations to gain insights into the mechanism of action of the Bacillus subtilis expansin BsEXLX1. We show that BsEXLX1 can slide on the hydrophobic surface of crystalline cellulose via the flat aromatic surface of its binding domain D2, comprised mainly of residues Trp125 and Trp126. Also, we observe that BsEXLX1 can hydrogen bond a free glucan chain in a twisted conformation and that the twisting is chiefly induced by means of residue Asp82 located on D1, which has been shown to be essential for expansin activity. These results suggest that BsEXLX1 could move on the surface of cellulose and disrupt hydrogen bonds by twisting glucan chains. Simulations of the inactive BsEXLX1 mutants Asp82Asn and Tyr73Ala indicate structural alterations around the twisting center in the domain D1, which suggest a molecular basis for the lack of activity of these mutants and corroborate the idea that BsEXLX1 works by inducing twists on glucan chains. Moreover, simulations of the double mutant Asp82Asn/Tyr73Leu predict the recovery of the lost activity of BsEXLX1-Asp82Asn. Our results provide a dynamical view of the expansin-substrate interactions at the molecular scale and help shed light on the expansin mechanism.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas de Bactérias/genética , Celulose/química , Celulose/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Conformação Proteica , Propriedades de SuperfícieRESUMO
The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the ß-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the ß-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.
Assuntos
Hipoglicemiantes/farmacologia , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Aumento de Peso , Células 3T3-L1 , Animais , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacocinética , Ligantes , Camundongos , Células NIH 3T3 , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Estrutura Secundária de Proteína , Tiazolidinedionas/química , Tiazolidinedionas/farmacocinética , Células U937RESUMO
Efficient conversion of lignocellulosic biomass to second-generation biofuels and valuable chemicals requires decomposition of resilient plant cell wall structure. Cell wall recalcitrance varies among plant species and even phenotypes, depending on the chemical composition of the noncellulosic matrix. Changing the amount and composition of branches attached to the hemicellulose backbone can significantly alter the cell wall strength and microstructure. We address the effect of hemicellulose composition on primary cell wall assembly forces by using the 3D-RISM-KH molecular theory of solvation, which provides statistical-mechanical sampling and molecular picture of hemicellulose arrangement around cellulose. We show that hemicellulose branches of arabinose, glucuronic acid, and especially glucuronate strengthen the primary cell wall by strongly coordinating to hydrogen bond donor sites on the cellulose surface. We reveal molecular forces maintaining the cell wall structure and provide directions for genetic modulation of plants and pretreatment design to render biomass more amenable to processing.
Assuntos
Biomassa , Parede Celular/química , Plantas/química , Polissacarídeos/química , Arabinose/química , Ácido Glucurônico/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Nanotecnologia , TermodinâmicaRESUMO
The peroxisome proliferator-activated receptor γ (PPARγ) is a target for treatment of type II diabetes and other conditions. PPARγ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPARγ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPARγ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPARγ target genes in 3T3-L1 cells (LPL, ORL1, and CEBPα) and PPARγ-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPARγ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPARγ LBD conformer and occupies a space near the ß-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPARγ, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Ω-loop among H2', H3, and the ß-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPARγ-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPARγ modulators.
Assuntos
Luteolina/farmacologia , PPAR gama/agonistas , Células 3T3-L1 , Animais , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Luteolina/química , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ácido Mirístico/química , PPAR gama/química , PPAR gama/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Tiazolidinedionas/antagonistas & inibidores , Tiazolidinedionas/farmacologiaRESUMO
Serine hydrolases cleave peptide and ester bonds and are ubiquitous in nature, with applications in biotechnology, in materials, and as drug targets. The serine hydrolase two-step mechanism employs a serine-histidine-aspartate/glutamate catalytic triad, where the histidine residue acts as a base to activate poor nucleophiles (a serine residue or a water molecule) and as an acid to allow the dissociation of poor leaving groups. This mechanism has been the subject of debate regarding how histidine shuttles the proton from the nucleophile to the leaving group. To elucidate the reaction mechanism of serine hydrolases, we employ quantum mechanics/molecular mechanics-based transition path sampling to obtain the reaction coordinate using the Aspergillus niger feruloyl esterase A (AnFaeA) as a model enzyme. The optimal reaction coordinates include terms involving nucleophilic attack on the carbonyl carbon and proton transfer to, and dissociation of, the leaving group. During the reaction, the histidine residue undergoes a reorientation on the time scale of hundreds of femtoseconds that supports the "moving histidine" mechanism, thus calling into question the "ring flip" mechanism. We find a concerted mechanism, where the transition state coincides with the tetrahedral intermediate with the histidine residue pointed between the nucleophile and the leaving group. Moreover, motions of the catalytic aspartate toward the histidine occur concertedly with proton abstraction by the catalytic histidine and help stabilize the transition state, thus partially explaining how serine hydrolases enable poor nucleophiles to attack the substrate carbonyl carbon. Rate calculations indicate that the second step (deacylation) is rate-determining, with a calculated rate constant of 66 s-1. Overall, these results reveal the pivotal role of active-site dynamics in the catalytic mechanism of AnFaeA, which is likely similar in other serine hydrolases.
Assuntos
Hidrolases de Éster Carboxílico , Hidrolases , Hidrolases de Éster Carboxílico/genética , Catálise , Estudos de AmostragemRESUMO
The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of ß-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the "Newton's cradle" catalytic mechanism recently proposed for this subfamily.
Assuntos
Celulase/química , Celulase/metabolismo , Phanerochaete/enzimologia , Catálise , Biologia Computacional , Cristalografia por Raios X , Ensaios Enzimáticos , Simulação de Dinâmica MolecularRESUMO
Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion.
Assuntos
Actinobacteria/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Lignina/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Oxirredução , Oxirredutases O-Desmetilantes/química , Multimerização Proteica , Especificidade por SubstratoRESUMO
Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.
Assuntos
Modelos Moleculares , PPAR gama/química , Conformação Proteica , Receptor X Retinoide alfa/química , Regulação Alostérica , Sítio Alostérico , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , PPAR gama/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Relação Quantitativa Estrutura-Atividade , Receptor X Retinoide alfa/metabolismoRESUMO
Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.
Assuntos
Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Celulose/análogos & derivados , Celulose/metabolismo , Sequência Consenso , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Movimento (Física) , Mutagênese Sítio-Dirigida , Filogenia , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tetroses/metabolismo , Difração de Raios XRESUMO
Enzymatic conversion of lignocellulosic biomass into biofuels and chemicals constitutes a potential route for sustainable development. Cellobiohydrolases are key enzymes used in industrial cocktails for depolymerization of crystalline cellulose, and their mechanism of action has been intensely studied in the past several years. Provided with a tunnel-like substrate-binding cavity, cellobiohydrolases possess the ability to processively hydrolyze glycosidic bonds of crystalline cellulose, yielding one molecule of cellobiose per catalytic cycle. As such, cellobiose expulsion from the product binding site is a necessary step in order to allow for the processive hydrolysis mechanism. However, the high-affinity binding of cellobiose to the enzyme impairs the process and causes activity inhibition due to reaction products. Here, we use molecular dynamics simulations to study the binding of cellobiose to the Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase and the effects of mutations that reduce cellobiose binding, without affecting the structural and dynamical integrities of the enzyme. We observe that the product binding site exhibits an intrinsic flexibility that can sterically hinder cellobiose release. Several point mutations in the product binding site reduce cellobiose-enzyme interactions, but not all modifications are able to maintain the structural integrity of the enzyme. In particular, mutation of charged residues in the TrCel7A product binding site causes perturbations that affect the structure of the loops that form the substrate-binding tunnel of the enzyme and, hence, may affect TrCel7A function in other steps of the hydrolysis mechanism. Our results suggest there is a trade-off between product inhibition and catalytic efficiency, and they provide directions for cellulases engineering.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Simulação de Dinâmica Molecular , Mutação , Sítios de Ligação , Celobiose/química , Probabilidade , Conformação Proteica , TrichodermaRESUMO
Plant biomass recalcitrance, a major obstacle to achieving sustainable production of second generation biofuels, arises mainly from the amorphous cell-wall matrix containing lignin and hemicellulose assembled into a complex supramolecular network that coats the cellulose fibrils. We employed the statistical-mechanical, 3D reference interaction site model with the Kovalenko-Hirata closure approximation (or 3D-RISM-KH molecular theory of solvation) to reveal the supramolecular interactions in this network and provide molecular-level insight into the effective lignin-lignin and lignin-hemicellulose thermodynamic interactions. We found that such interactions are hydrophobic and entropy-driven, and arise from the expelling of water from the mutual interaction surfaces. The molecular origin of these interactions is carbohydrate-π and π-π stacking forces, whose strengths are dependent on the lignin chemical composition. Methoxy substituents in the phenyl groups of lignin promote substantial entropic stabilization of the ligno-hemicellulosic matrix. Our results provide a detailed molecular view of the fundamental interactions within the secondary plant cell walls that lead to recalcitrance.
Assuntos
Parede Celular/química , Lignina/química , Modelos Biológicos , Plantas/química , Biomassa , SolubilidadeRESUMO
Plant biomass holds a promise for the production of second-generation ethanol via enzymatic hydrolysis, but its utilization as a biofuel resource is currently limited to a large extent by the cost and low efficiency of the cellulolytic enzymes. Considerable efforts have been dedicated to elucidate the mechanisms of the enzymatic process. It is well known that most cellulases possess a catalytic core domain and a carbohydrate binding module (CBM), without which the enzymatic activity can be drastically reduced. However, Cel12A members of the glycosyl hydrolases family 12 (GHF12) do not bear a CBM and yet are able to hydrolyze amorphous cellulose quite efficiently. Here, we use X-ray crystallography and molecular dynamics simulations to unravel the molecular basis underlying the catalytic capability of endoglucanase 3 from Trichoderma harzianum (ThEG3), a member of the GHF12 enzymes that lacks a CBM. A comparative analysis with the Cellulomonas fimi CBM identifies important residues mediating interactions of EG3s with amorphous regions of the cellulose. For instance, three aromatic residues constitute a harboring wall of hydrophobic contacts with the substrate in both ThEG3 and CfCBM structures. Moreover, residues at the entrance of the active site cleft of ThEG3 are identified, which might hydrogen bond to the substrate. We advocate that the ThEG3 residues Asn152 and Glu201 interact with the substrate similarly to the corresponding CfCBM residues Asn81 and Arg75. Altogether, these results show that CBM motifs are incorporated within the ThEG3 catalytic domain and suggest that the enzymatic efficiency is associated with the length and position of the substrate chain, being higher when the substrate interact with the aromatic residues at the entrance of the cleft and the catalytic triad. Our results provide guidelines for rational protein engineering aiming to improve interactions of GHF12 enzymes with cellulosic substrates.