Machine learning-aided engineering of a cytochrome P450 for optimal bioconversion of lignin fragments.

Using machine learning, molecular dynamics simulations, and density functional theory calculations we gain insight into the selectivity patterns of substrate activation by the cytochromes P450. In nature, the reactions catalyzed by the P450s lead to the biodegradation of xenobiotics, but recent work has shown that fungi utilize P450s for the activation of lignin fragments, such as monomer and dimer units. These fragments often are the building blocks of valuable materials, including drug molecules and fragrances, hence a highly selective biocatalyst that can produce these compounds in good yield with high selectivity would be an important step in biotechnology. In this work a detailed computational study is reported on two reaction channels of two P450 isozymes, namely the O-deethylation of guaethol by CYP255A and the O-demethylation versus aromatic hydroxylation of p-anisic acid by CYP199A4. The studies show that the second-coordination sphere plays a major role in substrate binding and positioning, heme access, and in the selectivity patterns. Moreover, the local environment affects the kinetics of the reaction through lowering or raising barrier heights. Furthermore, we predict a site-selective mutation for highly specific reaction channels for CYP199A4.

Biotransformation of Bisphenol by Human Cytochrome P450 2C9 Enzymes: A Density Functional Theory Study.

Bisphenol A (BPA, 2,2-bis-(4-hydroxyphenyl)propane) is used as a precursor in the synthesis of polycarbonate and epoxy plastics; however, its availability in the environment is causing toxicity as an endocrine-disrupting chemical. Metabolism of BPA and their analogues (substitutes) is generally performed by liver cytochrome P450 enzymes and often leads to a mixture of products, and some of those are toxic. To understand the product distributions of P450 activation of BPA, we have performed a computational study into the mechanisms and reactivities using large model structures of a human P450 isozyme (P450 2C9) with BPA bound. Density functional theory (DFT) calculations on mechanisms of BPA activation by a P450 compound I model were investigated, leading to a number of possible products. The substrate-binding pocket is tight, and as a consequence, aliphatic hydroxylation is not feasible as the methyl substituents of BPA cannot reach compound I well due to constraints of the substrate-binding pocket. Instead, we find low-energy pathways that are initiated with phenol hydrogen atom abstraction followed by OH rebound to the phenolic ortho- or para-position. The barriers of para-rebound are well lower in energy than those for ortho-rebound, and consequently, our P450 2C9 model predicts dominant hydroxycumyl alcohol products. The reactions proceed through two-state reactivity on competing doublet and quartet spin state surfaces. The calculations show fast and efficient substrate activation on a doublet spin state surface with a rate-determining electrophilic addition step, while the quartet spin state surface has multiple high-energy barriers that can also lead to various side products including C4-aromatic hydroxylation. This work shows that product formation is more feasible on the low spin state, while the physicochemical properties of the substrate govern barrier heights of the rate-determining step of the reaction. Finally, the importance of the second-coordination sphere is highlighted that determines the product distributions and guides the bifurcation pathways.

MassCCS: A High-Performance Collision Cross-Section Software for Large Macromolecular Assemblies.

Ion mobility mass spectrometry (IM-MS) techniques have become highly valued as a tool for structural characterization of biomolecular systems since they yield accurate measurements of the rotationally averaged collision cross-section (CCS) against a buffer gas. Despite its enormous potential, IM-MS data interpretation is often challenging due to the conformational isomerism of metabolites, lipids, proteins, and other biomolecules in the gas phase. Therefore, reliable and fast CCS calculations are needed to help interpret IM-MS data. In this work, we present MassCCS, a parallelized open-source code for computing CCS of molecules ranging from small organic compounds to massive protein assemblies at the trajectory method level of description using atomic and molecular buffer gas particles. The performance of the code is comparable to other available software for small molecules and proteins but is significantly faster for larger macromolecular assemblies. We performed extensive tests regarding accuracy, performance, and scalability with system size and number of CPU cores. MassCCS has proven highly accurate and efficient, with execution times under a few minutes, even for large (84.87 MDa) virus capsid assemblies with very modest computational resources. MassCCS is freely available at https://github.com/cces-cepid/massccs.

Insights into the dual cleavage activity of the GH16 laminarinase enzyme class on ß-1,3 and ß-1,4 glycosidic bonds.

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.

Structural insights into ß-1,3-glucan cleavage by a glycoside hydrolase family.

The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.

Publisher Correction: Structural insights into ß-1,3-glucan cleavage by a glycoside hydrolase family.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nanoscale Wetting of Crystalline Cellulose.

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 (11̅0), Iß (010), and Iß (110) faces, as well as the faces of human-made celluloses II and III_I (100), (11̅0), (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 (11̅0) 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.

Importance of Water in Maintaining Softwood Secondary Cell Wall Nanostructure.

Water is one of the principal constituents by mass of living plant cell walls. However, its role and interactions with secondary cell wall polysaccharides and the impact of dehydration and subsequent rehydration on the molecular architecture are still to be elucidated. This work combines multidimensional solid-state 13C magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) with molecular dynamics modeling to decipher the role of water in the molecular architecture of softwood secondary cell walls. The proximities between all main polymers, their molecular conformations, and interaction energies are compared in never-dried, oven-dried, and rehydrated states. Water is shown to play a critical role at the hemicellulose-cellulose interface. After significant molecular shrinkage caused by dehydration, the original molecular conformation is not fully recovered after rehydration. The changes include xylan becoming more closely and irreversibly associated with cellulose and some mannan becoming more mobile and changing conformation. These irreversible nanostructural changes provide a basis for explaining and improving the properties of wood-based materials.

QM/MM Simulations of Enzymatic Hydrolysis of Cellulose: Probing the Viability of an Endocyclic Mechanism for an Inverting Cellulase.

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.

Characterization and engineering of a plastic-degrading aromatic polyesterase.

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.

Adsorption of CO2 and CH4 in MIL-47 investigated by the 3D-RISM molecular theory of solvation.

Metal-organic frameworks (MOFs) comprise a class of highly porous nanomaterials formed by the assembly of organic molecular templates connected by metal ions. These materials exhibit a large diversity of pore size and geometry, topology, surface area, and chemical functionality. MOFs are particularly promising materials for developing new technologies for capture and storage of greenhouse gases such as methane and carbon dioxide. Here we apply the three dimensional reference interaction site model (3D-RISM) molecular theory of solvation to study the interactions of CO2 and CH4 with the metal-organic material MIL-47. The 3D-RISM integral equations were solved to determine the three dimensional density correlation functions of the gas (solvent) relative to the atomic positions of the MIL-47 framework, treated as static solute sites. The computed solvent spatial distributions inside MIL-47 pores were used to identify whether or not there exist preferable binding sites and the binding free energy landscape for the gas of interest at low computational costs compared with other molecular modeling techniques, such as grand canonical Monte Carlo and molecular dynamics simulations. The 3D-RISM formalism was applied to pure CO2, pure CH4, and binary mixtures of these gases of various compositions under different pressure conditions. The results indicate that both gases bind very weakly to MIL-47 and that this material exhibits nearly vanishing CO2/CH4 selectivity. The 3D-RISM computations presented here can be extended to investigate the physical adsorption of gases on other MOFs and nanoporous materials, providing an alternative low-cost computational approach to study gas capture and storage in nanoporous materials in general and, in particular, to determine the binding free-energy landscape in these systems.

The Patterned Structure of Galactoglucomannan Suggests It May Bind to Cellulose in Seed Mucilage.

The interaction between mannan polysaccharides and cellulose microfibrils contributes to cell wall properties in some vascular plants, but the molecular arrangement of mannan in the cell wall and the nature of the molecular bonding between mannan and cellulose remain unknown. Previous studies have shown that mannan is important in maintaining Arabidopsis (Arabidopsis thaliana) seed mucilage architecture, and that Cellulose Synthase-Like A2 (CSLA2) synthesizes a glucomannan backbone, which Mannan α-Galactosyl Transferase1 (MAGT1/GlycosylTransferase-Like6/Mucilage Related10) might decorate with single α-Gal branches. Here, we investigated the ratio and sequence of Man and Glc and the arrangement of Gal residues in Arabidopsis mucilage mannan using enzyme sequential digestion, carbohydrate gel electrophoresis, and mass spectrometry. We found that seed mucilage galactoglucomannan has a backbone consisting of the repeating disaccharide [4)-ß-Glc-(1,4)-ß-Man-(1,], and most of the Man residues in the backbone are substituted by single α-1,6-Gal. CSLA2 is responsible for the synthesis of this patterned glucomannan backbone and MAGT1 catalyses the addition of α-Gal. In vitro activity assays revealed that MAGT1 transferred α-Gal from UDP-Gal only to Man residues within the CSLA2 patterned glucomannan backbone acceptor. These results indicate that CSLAs and galactosyltransferases are able to make precisely defined galactoglucomannan structures. Molecular dynamics simulations suggested this patterned galactoglucomannan is able to bind stably to some hydrophilic faces and to hydrophobic faces of cellulose microfibrils. A specialization of the biosynthetic machinery to make galactoglucomannan with a patterned structure may therefore regulate the mode of binding of this hemicellulose to cellulose fibrils.

High performance collision cross section calculation-HPCCS.

Since the commercial introduction of Ion Mobility coupled with Mass Spectrometry (IM-MS) devices in 2003, a large number of research laboratories have embraced the technique. IM-MS is a fairly rapid experiment used as a molecular separation tool and to obtain structural information. The interpretation of IM-MS data is still challenging and relies heavily on theoretical calculations of the molecule's collision cross section (CCS) against a buffer gas. Here, a new software (HPCCS) is presented, which performs CCS calculations using high perfomance computing techniques. Based on the trajectory method, HPCCS can accurately calculate CCS for a great variety of molecules, ranging from small organic molecules to large protein complexes, using helium or nitrogen as buffer gas with considerable gains in computer time compared to publicly available codes under the same level of theory. HPCCS is available as free software under the Academic Use License at https://github.com/cepid-cces/hpccs. © 2018 Wiley Periodicals, Inc.

Concerted motions and large-scale structural fluctuations of Trichoderma reesei Cel7A cellobiohydrolase.

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.

Structural and functional characterization of a highly secreted α-l-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse.

Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. α-l-Arabinofuranosidases that belong to the Glycoside Hydrolase family 62 (GH62) have important applications in biofuel production from plant biomass by hydrolyzing arabinoxylans, found in both the primary and secondary cell walls of plants. In this work, we identified a GH62 α-l-arabinofuranosidase (AnAbf62Awt) that was highly secreted when Aspergillus nidulans was cultivated on sugarcane bagasse. The gene AN7908 was cloned and transformed in A. nidulans for homologous production of AnAbf62Awt, and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coli and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62Awt and the non-glycosylated enzyme from E. coli (AnAbf62Adeglyc) were highly similar. In addition, the designed glycomutant AnAbf62AN83Q presented similar patterns of secretion and activity to the AnAbf62Awt, indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62Adeglyc was obtained and the 1.7Å resolution model showed a five-bladed ß-propeller fold, which is conserved in family GH62. Mutants AnAbf62AY312F and AnAbf62AY312S showed that Y312 was an important substrate-binding residue. Molecular dynamics simulations indicated that the loop containing Y312 could access different conformations separated by moderately low energy barriers. One of these conformations, comprising a local minimum, is responsible for placing Y312 in the vicinity of the arabinose glycosidic bond, and thus, may be important for catalytic efficiency.

Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms: Implications for Xylan Interaction with Cellulose.

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.

Effects of Xylan Side-Chain Substitutions on Xylan-Cellulose Interactions and Implications for Thermal Pretreatment of Cellulosic Biomass.

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.

Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases.

Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and ß-glucan chains. Several structural and functional studies involving GH12 enzymes tried to explain the main patterns of xyloglucan activity, but what really determines xyloglucanase specificity remains elusive. Here, three fungal GH12 enzymes from Aspergillus clavatus (AclaXegA), A. zonatus (AspzoGH12), and A. terreus (AtEglD) were studied to unveil the molecular basis for substrate specificity. Using functional assays, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that three main regions are responsible for substrate selectivity: (i) the YSG group in loop 1; (ii) the SST group in loop 2; and (iii) loop A3-B3 and neighboring residues. Functional assays and sequence alignment showed that while AclaXegA is specific to xyloglucan, AtEglD cleaves ß-glucan, and xyloglucan. However, AspzoGH12 was also shown to be promiscuous contrarily to a sequence alignment-based prediction. We find that residues Y111 and R93 in AtEglD harbor the substrate in an adequate orientation for hydrolysis in the catalytic cleft entrance and that residues Y19 in AclaXegA and Y30 in AspzoGH12 partially compensate the absence of the YSG segment, typically found in promiscuous enzymes. The results point out the multiple structural factors underlying the substrate specificity of GH12 enzymes. Biotechnol. Bioeng. 2016;113: 2577-2586. © 2016 Wiley Periodicals, Inc.

Cellulose Aggregation under Hydrothermal Pretreatment Conditions.

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.

Molecular dynamics of the Bacillus subtilis expansin EXLX1: interaction with substrates and structural basis of the lack of activity of mutants.

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.