Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

Redesigned Hybrid Nylons with Optical Clarity and Chemical Recyclability.

Aliphatic polyamides, or nylons, are typically highly crystalline and thermally robust polymers used in high-performance applications. Nylon 6, a high-ceiling-temperature (HCT) polyamide from ε-caprolactam, lacks expedient chemical recyclability, while low-ceiling temperature (LCT) nylon 4 from pyrrolidone exhibits complete chemical recyclability, but it is thermally unstable and not melt-processable. Here, we introduce a hybrid nylon, nylon 4/6, based on a bicyclic lactam composed of both HCT ε-caprolactam and LCT pyrrolidone motifs in a hybridized offspring structure. Hybrid nylon 4/6 overcomes trade-offs in (de)polymerizability and performance properties of the parent nylons, exhibiting both excellent polymerization and facile depolymerization characteristics. This stereoregular polyamide forms nanocrystalline domains, allowing optical clarity and high thermal stability, however, without displaying a melting transition before decomposition. Of a series of statistical copolymers comprising nylon 4/6 and nylon 4, a 50/50 copolymer achieves the greatest synergy in both reactivity and polymer properties of each homopolymer, offering an amorphous nylon with favorable properties, including optical clarity, a high glass transition temperature, melt processability, and full chemical recyclability.

A temperature-sensitive FERONIA mutant allele that alters root hair growth.

In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.

Advantages of a distant cellulase catalytic base.

The inverting glycoside hydrolase Trichoderma reesei (Hypocrea jecorina) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle.

Multiscale Kinetic Modeling Reveals an Ensemble of Cl-/H+ Exchange Pathways in ClC-ec1 Antiporter.

Despite several years of research, the ion exchange mechanisms in chloride/proton antiporters and many other coupled transporters are not yet understood at the molecular level. Here, we present a novel approach to kinetic modeling and apply it to ion exchange in ClC-ec1. Our multiscale kinetic model is developed by (1) calculating the state-to-state rate coefficients with reactive and polarizable molecular dynamics simulations, (2) optimizing these rates in a global kinetic network, and (3) predicting new electrophysiological results. The model shows that the robust Cl:H exchange ratio (2.2:1) can indeed arise from kinetic coupling without large protein conformational changes, indicating a possible facile evolutionary connection to chloride channels. The E148 amino acid residue is shown to couple chloride and proton transport through protonation-dependent blockage of the central anion binding site and an anion-dependent pKa value, which influences proton transport. The results demonstrate how an ensemble of different exchange pathways, as opposed to a single series of transitions, culminates in the macroscopic observables of the antiporter, such as transport rates, chloride/proton stoichiometry, and pH dependence.

The Origin of Coupled Chloride and Proton Transport in a Cl-/H+ Antiporter.

The ClC family of transmembrane proteins functions throughout nature to control the transport of Cl- ions across biological membranes. ClC-ec1 from Escherichia coli is an antiporter, coupling the transport of Cl- and H+ ions in opposite directions and driven by the concentration gradients of the ions. Despite keen interest in this protein, the molecular mechanism of the Cl-/H+ coupling has not been fully elucidated. Here, we have used multiscale simulation to help identify the essential mechanism of the Cl-/H+ coupling. We find that the highest barrier for proton transport (PT) from the intra- to extracellular solution is attributable to a chemical reaction, the deprotonation of glutamic acid 148 (E148). This barrier is significantly reduced by the binding of Cl- in the "central" site (Cl-cen), which displaces E148 and thereby facilitates its deprotonation. Conversely, in the absence of Cl-cen E148 favors the "down" conformation, which results in a much higher cumulative rotation and deprotonation barrier that effectively blocks PT to the extracellular solution. Thus, the rotation of E148 plays a critical role in defining the Cl-/H+ coupling. As a control, we have also simulated PT in the ClC-ec1 E148A mutant to further understand the role of this residue. Replacement with a non-protonatable residue greatly increases the free energy barrier for PT from E203 to the extracellular solution, explaining the experimental result that PT in E148A is blocked whether or not Cl-cen is present. The results presented here suggest both how a chemical reaction can control the rate of PT and also how it can provide a mechanism for a coupling of the two ion transport processes.

How sugars pucker: electronic structure calculations map the kinetic landscape of five biologically paramount monosaccharides and their implications for enzymatic catalysis.

Glycoside hydrolases (GHs) distort carbohydrate ring geometry along particular "catalytic itineraries" during the cleavage of glycosidic bonds, illustrating the relationship between substrate conformation and reactivity. Previous theoretical studies of thermodynamics of isolated monosaccharides offer insights into the catalytic itineraries of particular sugars. However, kinetic accessibility of carbohydrate puckering conformations and the role of exocyclic groups have not yet been thoroughly addressed. Here we present the first complete library of low-energy local minima and puckering interconversion transition states for five biologically relevant pyranose sugars: ß-xylose, ß-mannose, α-glucose, ß-glucose, and ß-N-acetylglucosamine. These were obtained by a thorough theoretical investigation each of the 38 IUPAC designated puckering geometries and all possible conformations of the exocyclic groups. These calculations demonstrate that exocyclic groups must be explicitly considered when examining these interconversion pathways. Furthermore, these data enable evaluation of previous hypotheses of why enzymes perturb ring geometries from the low-energy equatorial chair ((4)C1) conformation. They show that the relative thermodynamics alone do not universally correlate with GH catalytic itineraries. For some sugars, particular puckers offer both catalytically favorable electronic structure properties, such as anomeric carbon partial charge, and low kinetic barriers to achieve a given puckering conformation. However, different factors correlate with catalytic itineraries for other sugars; for ß-N-acetylglucosamine, the key N-acetyl arm confounds the puckering landscape and appears to be the crucial factor. Overall, this study reveals a more comprehensive understanding of why particular puckering geometries are favored in carbohydrate catalysis concomitant with the complexity of glycobiology.

The reaction mechanism of the Ideonella sakaiensis PETase enzyme.

Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion.

ATESA: An Automated Aimless Shooting Workflow.

Transition path sampling methods are powerful tools for studying the dynamics of rare events in molecular simulations. However, these methods are generally restricted to experts with the knowledge and resources to properly set up and analyze the often hundreds of thousands of simulations that constitute a complete study. Aimless Transition Ensemble Sampling and Analysis (ATESA) is a new open-source software program written in Python that automates a full transition path sampling workflow based on the aimless shooting algorithm, streamlining the process and reducing the barrier to use for researchers new to this approach. This introduction to ATESA includes a demonstration of a complete transition path sampling process flow for an example reaction, including finding an initial transition state, sampling with aimless shooting, building a reaction coordinate with inertial likelihood maximization, verifying that coordinate with committor analysis, and measuring the reaction energy profile with umbrella sampling. We also describe our implementation of a termination criterion for aimless shooting based on the Godambe information calculated during model building with likelihood maximization as well as a novel approach to constraining simulations to the desired rare event pathway during umbrella sampling.

Energy-Resolved Mass Spectrometry as a Tool for Identification of Lignin Depolymerization Products.

Lignin is the largest source of bio-based aromatic compounds in nature, and its valorization is essential to the sustainability of lignocellulosic biorefining. Characterizing lignin-derived compounds remains challenging due to the heterogeneity of this biopolymer. Tandem mass spectrometry is a promising tool for lignin structural analytics, as fragmentation patterns of model compounds can be extrapolated to identify characteristic moieties in complex samples. This work extended previous resonance excitation-type collision-induced dissociation (CID) methods that identified lignin oligomers containing ß-O-4, ß-5, and ß-ß bonds, to also identify characteristics of 5-5, ß-1, and 4-O-5 dimers, enabled by quadrupole time-of-flight (QTOF) CID with energy-resolved mass spectrometry (ERMS). Overall, QTOF-ERMS offers in-depth structural information and could ultimately contribute to tools for high-throughput lignin dimer identification.

Ni- and Ni/Pd-Catalyzed Reductive Coupling of Lignin-Derived Aromatics to Access Biobased Plasticizers.

Lignin-derived aromatic chemicals offer a compelling alternative to petrochemical feedstocks, and new applications are the focus of extensive interest. 4-Hydroxybenzoic acid (H), vanillic acid (G), and syringic acid (S) are readily obtained via oxidative depolymerization of hardwood lignin substrates. Here, we explore the use of these compounds to access biaryl dicarboxylate esters that represent biobased, less toxic alternatives to phthalate plasticizers. Chemical and electrochemical methods are developed for catalytic reductive coupling of sulfonate derivatives of H, G, and S to access all possible homo- and cross-coupling products. A conventional NiCl2/bipyridine catalyst is able to access the H-H and G-G products, but new catalysts are identified to afford the more challenging coupling products, including a NiCl2/bisphosphine catalyst for S-S and a NiCl2/phenanthroline/PdCl2/phosphine cocatalyst system for H-G, H-S, and G-S. High-throughput experimentation methods with a chemical reductant (Zn powder) are shown to provide an efficient screening platform for identification of new catalysts, while electrochemical methods can access improved yields and/or facilitate implementation on larger scale. Plasticizer tests are performed with poly(vinyl chloride), using esters of the 4,4'-biaryl dicarboxylate products. The H-G and G-G derivatives, in particular, exhibit performance advantages relative to an established petroleum-based phthalate ester plasticizer.

Unraveling the reactions that unravel cellulose.

For over 90 years, researchers have postulated mechanisms for the cleavage of cellulose's glycosidic bonds and resulting formation of levoglucosan without reaching consensus. These reactions are key primary reactions in thermal processes for the production of carbon-neutral, renewable transportation fuels. Previous literature reports have proposed a variety of mainly heterolytic and homolytic mechanisms, but there has been insufficient evidence to settle the debate. Using density functional theory (DFT) methods and implicit solvent, we compared the likelihood of forming either radical or ionic intermediates. We discovered a concerted reaction mechanism that is more favorable than previously proposed mechanisms and is in better alignment with experimental findings. This new understanding of the mechanism of cellulose thermal decomposition opens the door to accurate process modeling and educated catalyst design, which are vital steps toward producing more cost-efficient renewable energy.

Click-Chemistry-Based Free Azide versus Azido Sugar Detection Enables Rapid In Vivo Screening of Glycosynthase Activity.

Engineering of carbohydrate-active enzymes such as glycosynthases to enable chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable ultrahigh-throughput screening methods capable of robustly detecting either starting substrates or end-products of the glycosidic bond formation reaction. Currently, there are limited screening methods available for rapid and highly sensitive single-cell-based screening of glycosynthase enzymes employing azido sugars as activated donor glycosyl substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides as substrates versus free inorganic azides as reaction products that facilitated an ultrahigh-throughput in vivo single-cell-based assay of glycosynthase activity. This assay was developed based on the distinct differences observed in relative fluorescence intensity of the triazole-containing fluorophore product formed during the click-chemistry reaction of organic glycosyl azides versus inorganic azides. This discovery formed the basis for proof of concept validation of a directed evolution methodology for screening and sorting glycosynthase mutants capable of synthesis of targeted fucosylated oligosaccharides. Our screening approach facilitated fluorescence-activated cell sorting of an error-prone polymerase chain reaction-based mutant library of fucosynthases expressed in Escherichia coli to identify several novel mutants that showed increased activity for ß-fucosyl azide-activated donor sugars toward desired acceptor sugars (e.g., pNP-xylose and lactose). Finally, we discuss avenues for improving this proof of concept in vivo assay method to identify better glycosynthase mutants and further demonstrate the broader applicability of this screening methodology for synthesis of bespoke glycans.

Best Practices for Foundations in Molecular Simulations [Article v1.0].

This document provides a starting point for approaching molecular simulations, guiding beginning practitioners to what issues they need to know about before and while starting their first simulations, and why those issues are so critical. This document makes no claims to provide an adequate introduction to the subject on its own. Instead, our goal is to help people know what issues are critical before beginning, and to provide references to good resources on those topics. We also provide a checklist of key issues to consider before and while setting up molecular simulations which may serve as a foundation for other best practices documents.

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases.

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase (TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleaving the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2SO ring configuration as it reaches its binding site. This work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.

Sodium ion interactions with aqueous glucose: insights from quantum mechanics, molecular dynamics, and experiment.

In the last several decades, significant efforts have been conducted to understand the fundamental reactivity of glucose derived from plant biomass in various chemical environments for conversion to renewable fuels and chemicals. For reactions of glucose in water, it is known that inorganic salts naturally present in biomass alter the product distribution in various deconstruction processes. However, the molecular-level interactions of alkali metal ions and glucose are unknown. These interactions are of physiological interest as well, for example, as they relate to cation-glucose cotransport. Here, we employ quantum mechanics (QM) to understand the interaction of a prevalent alkali metal, sodium, with glucose from a structural and thermodynamic perspective. The effect on ß-glucose is subtle: a sodium ion perturbs bond lengths and atomic partial charges less than rotating a hydroxymethyl group. In contrast, the presence of a sodium ion significantly perturbs the partial charges of α-glucose anomeric and ring oxygens. Molecular dynamics (MD) simulations provide dynamic sampling in explicit water, and both the QM and the MD results show that sodium ions associate at many positions with respect to glucose with reasonably equivalent propensity. This promiscuous binding nature of Na(+) suggests that computational studies of glucose reactions in the presence of inorganic salts need to ensure thorough sampling of the cation positions, in addition to sampling glucose rotamers. The effect of NaCl on the relative populations of the anomers is experimentally quantified with light polarimetry. These results support the computational findings that Na(+) interacts similarly with α- and ß-glucose.