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Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

BackgroundGlycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions.ResultsSingle, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss of specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides.ConclusionsThe identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.

Stilbenes are diphenyl ethene compounds produced naturally in a wide variety of plant species and some bacteria. Stilbenes are also derived from lignin during kraft pulping. Stilbene cleavage oxygenases (SCOs) cleave the central double bond of stilbenes, forming two phenolic aldehydes. Here, we report the structure of an SCO. The X-ray structure of NOV1 from Novosphingobium aromaticivorans was determined in complex with its substrate resveratrol (1.89 Å), its product vanillin (1.75 Å), and without any bound ligand (1.61 Å). The enzyme is a seven-bladed β-propeller with an iron cofactor coordinated by four histidines. In all three structures, dioxygen is observed bound to the iron in a side-on fashion. These structures, along with EPR analysis, allow us to propose a mechanism in which a ferric-superoxide reacts with substrate activated by deprotonation of a phenol group at position 4 of the substrate, which allows movement of electron density toward the central double bond and thus facilitates reaction with the ferric superoxide electrophile. Correspondingly, NOV1 cleaves a wide range of other stilbene-like compounds with a 4'-OH group, offering potential in processing some solubilized fragments of lignin into monomer aromatic compounds.

BackgroundCarbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.ResultsCelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.ConclusionWe have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design.

Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.

With the recent successes in determining membrane protein structures, we explore the tractability of determining representatives for the entire human membrane proteome. This proteome contains 2,925 unique integral α-helical transmembrane domain sequences that cluster into 1,201 families sharing more than 25% sequence identity. Structures of 100 optimally selected targets would increase the fraction of modelable human α-helical transmembrane domains from 26% to 58%, thus providing structure/function information not otherwise available.

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs (1-4). Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity (5, 6). Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases (7). SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl- or palmitoyl-CoA (8, 9). The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome [b.sub.5], which regenerates the di-iron centre (10). To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome [b.sub.5] to the di-iron centre., A Δ2-23 amino-terminal truncation of mouse SCD1 was crystallized in lipidic cubic phase (11) and the structure was solved by single-wavelength anomalous dispersion (Extended Data Table 1). SCD1 has four [...]

DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A [[DELTA].sup.9] desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal [His.sub.6] or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ~10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O[sub.2], electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe[sup.2+]/Fe[sup.3+] species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the OO bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results., Author(s): Justin F. Acheson [1]; Lucas J. Bailey [1]; Thomas C. Brunold [2]; Brian G. Fox (corresponding author) [1] Aromatic rings are among the most widely distributed and recognizable forms [...]