Michael F. Crowley

The implementation of a fast and accurate QM/MM potential method in Amber

Version 9 of the Amber simulation programs includes a new semi‐empirical hybrid QM/MM functionality. This includes support for implicit solvent (generalized Born) and for periodic explicit solvent simulations using a newly developed QM/MM implementation of the particle mesh Ewald (PME) method. The code provides sufficiently accurate gradients to run constant energy QM/MM MD simulations for many nanoseconds. The link atom approach used for treating the QM/MM boundary shows improved performance, and the user interface has been rewritten to bring the format into line with classical MD simulations. Support is provided for the PM3, PDDG/PM3, PM3CARB1, AM1, MNDO, and PDDG/MNDO semi‐empirical Hamiltonians as well as the self‐consistent charge density functional tight binding (SCC‐DFTB) method. Performance has been improved to the point where using QM/MM, for a QM system of 71 atoms within an explicitly solvated protein using periodic boundaries and PME requires less than twice the cpu time of the corresponding classical simulation.

Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA.

An Enzyme Drill Cellulase enzymes degrade the cell walls of plants by breaking down cellulose into its constituent sugar fragments and thus have attracted interest for biofuels production. Using transmission electron microscopy Brunecky et al. (p. 1513; see the Perspective by Berlin) discovered that an especially active cellulase, CelA, from Caldicellulosiruptor bescii bacteria does not move along the surface of the substrate, but drills into the cellulose to form cavities. Electron microscopy reveals that a cellulose-degrading enzyme operates by drilling down, as well as by roaming the surface. [Also see Perspective by Berlin] Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldicellulosiruptor bescii uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo‐ and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that nature’s repertoire of cellulose digestion paradigms remain only partially discovered and understood.

Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.

Cellulose is a crystalline polymer of β1,4-D-glucose that is difficult to deconstruct to sugars by enzymes. The recalcitrance of cellulose microfibrils is a function of both the shape of cellulose microfibrils and the intrinsic work required to decrystallize individual chains, the latter of which is calculated here from the surfaces of four crystalline cellulose polymorphs: cellulose Iβ, cellulose Iα, cellulose II, and cellulose III(I). For edge chains, the order of decrystallization work is as follows (from highest to lowest): Iβ, Iα, ΙΙΙ(Ι), and II. For cellulose Iβ, we compare chains from three different locations on the surface and find that an increasing number of intralayer hydrogen bonds (from 0 to 2) increases the intrinsic decrystallization work. From these results, we propose a microkinetic model for the deconstruction of cellulose (and chitin) by processive enzymes, which when taken with a previous study [Horn et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18089] identifies the thermodynamic and kinetic attributes of enzyme and substrate engineering for enhanced cellulose (or chitin) conversion. Overall, this study provides new insights into the molecular interactions that form the structural basis of cellulose, which is the primary building block of plant cell walls, and highlights the need for experimentally determining microfibril shape at the nanometer length scale when comparing conversion rates of cellulose polymorphs by enzymes.

Applications of computational science for understanding enzymatic deconstruction of cellulose

Understanding the molecular-level mechanisms that enzymes employ to deconstruct plant cell walls is a fundamental scientific challenge with significant ramifications for renewable fuel production from biomass. In nature, bacteria and fungi use enzyme cocktails that include processive and non-processive cellulases and hemicellulases to convert cellulose and hemicellulose to soluble sugars. Catalyzed by an accelerated biofuels R&D portfolio, there is now a wealth of new structural and experimental insights related to cellulases and the structure of plant cell walls. From this background, computational approaches commonly used in other fields are now poised to offer insights complementary to experiments designed to probe mechanisms of plant cell wall deconstruction. Here we outline the current status of computational approaches for a collection of critical problems in cellulose deconstruction. We discuss path sampling methods to measure rates of elementary steps of enzyme action, coarse-grained modeling for understanding macromolecular, cellulosomal complexes, methods to screen for enzyme improvements, and studies of cellulose at the molecular level. Overall, simulation is a complementary tool to understand carbohydrate-active enzymes and plant cell walls, which will enable industrial processes for the production of advanced, renewable fuels.

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.

Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase Gh61D from the Basidiomycota Fungus Phanerochaete Chrysosporium

Background: Lytic polysaccharide monooxygenases (LPMOs) represent a recently discovered enzymatic route to cleave carbohydrates. Results: We report the first basidiomycete LPMO structure and describe enzyme-cellulose interactions with simulation. Conclusion: We characterize the copper-containing active site and identify loops important for substrate recognition and binding. Significance: This structure is the first LPMO from a model basidiomycete fungus that contains many LPMO genes. Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain.

Identification of amino acids responsible for processivity in a Family 1 carbohydrate-binding module from a fungal cellulase.

We probe the molecular-level behavior of the Family 1 carbohydrate-binding module (CBM) from a commonly studied fungal cellulase, the Family 7 cellobiohydrolase (Cel7A) from Trichoderma reesei, on the hydrophobic face of crystalline cellulose. With a fully atomistic model, we predict that the CBM alone exhibits regions of thermodynamic stability along a cellulose chain corresponding to a cellobiose unit, which is the catalytic product of the entire Cel7A enzyme. In addition, we determine which residues and the types of interactions that are responsible for the observed processivity length scale of the CBM: Y5, Q7, N29, and Y32. These results imply that the CBM can anchor the Cel7A enzyme at discrete points along a cellulose chain and thus aid in both recognizing cellulose chain ends for initial attachment to cellulose as well as aid in enzymatic catalysis by diffusing between stable wells on a length scale commensurate with the catalytic, processive cycle of Cel7A during cellulose hydrolysis. Comparison of other Family 1 CBMs show high functional homology to the four amino acids responsible for the processivity length scale on the surface of crystalline cellulose, which suggests that Family 1 CBMs may generally employ this type of approach for translation on the cellulose surface. Overall, this work provides further insight into the molecular-level mechanisms by which a CBM recognizes and interacts with cellulose.

A biophysical perspective on the cellulosome: new opportunities for biomass conversion

The cellulosome is a multiprotein complex, produced primarily by anaerobic microorganisms, which functions to degrade lignocellulosic materials. An important topic of current debate is whether cellulosomal systems display greater ability to deconstruct complex biomass materials (e.g. plant cell walls) than nonaggregated enzymes, and in so doing would be appropriate for improved, commercial bioconversion processes. To sufficiently understand the complex macromolecular processes between plant cell wall polymers, cellulolytic microbes, and their secreted enzymes, a highly concerted research approach is required. Adaptation of existing biophysical techniques and development of new science tools must be applied to this system. This review focuses on strategies likely to permit improved understanding of the bacterial cellulosome using biophysical approaches, with emphasis on advanced imaging and computational techniques.

Comparison of Cellulose Iβ Simulations with Three Carbohydrate Force Fields

Molecular dynamics simulations of cellulose have recently become more prevalent due to increased interest in renewable energy applications, and many atomistic and coarse-grained force fields exist that can be applied to cellulose. However, to date no systematic comparison between carbohydrate force fields has been conducted for this important system. To that end, we present a molecular dynamics simulation study of hydrated, 36-chain cellulose Iβ microfibrils at room temperature with three carbohydrate force fields (CHARMM35, GLYCAM06, and Gromos 45a4) up to the near-microsecond time scale. Our results indicate that each of these simulated microfibrils diverge from the cellulose Iβ crystal structure to varying degrees under the conditions tested. The CHARMM35 and GLYCAM06 force fields eventually result in structures similar to those observed at 500 K with the same force fields, which are consistent with the experimentally observed high-temperature behavior of cellulose I. The third force field, Gromos 45a4, produces behavior significantly different from experiment, from the other two force fields, and from previously reported simulations with this force field using shorter simulation times and constrained periodic boundary conditions. For the GLYCAM06 force field, initial hydrogen-bond conformations and choice of electrostatic scaling factors significantly affect the rate of structural divergence. Our results suggest dramatically different time scales for convergence of properties of interest, which is important in the design of computational studies and comparisons to experimental data. This study highlights that further experimental and theoretical work is required to understand the structure of small diameter cellulose microfibrils typical of plant cellulose.

Adventures in Improving the Scaling and Accuracy of a Parallel Molecular Dynamics Program

We report our work to parallelize the Particle Mesh Ewald (PME) method to compute the long-range electrostatic interactions in the molecular dynamics program AMBER and to extend the scalability of the PME method to hundreds of processors.