Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.

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 ß-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.

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin ß-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6.

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of ß-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. ß-Aryl ether units are typically abundant in lignin, corresponding to 50-70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic ß-aryl ether (ß-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the ß-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of ß-Aryl Ether Bonds in Lignin.

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via ß-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent ß-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because ß-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic ß-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS.

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.

Phylogenomically guided identification of industrially relevant GH1 ß-glucosidases through DNA synthesis and nanostructure-initiator mass spectrometry.

Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 ß-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different ß-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

Rapid kinetic characterization of glycosyl hydrolases based on oxime derivatization and nanostructure-initiator mass spectrometry (NIMS).

Glycoside hydrolases (GHs) are critical to cycling of plant biomass in the environment, digestion of complex polysaccharides by the human gut microbiome, and industrial activities such as deployment of cellulosic biofuels. High-throughput sequencing methods show tremendous sequence diversity among GHs, yet relatively few examples from the over 150,000 unique domain arrangements containing GHs have been functionally characterized. Here, we show how cell-free expression, bioconjugate chemistry, and surface-based mass spectrometry can be used to study glycoside hydrolase reactions with plant biomass. Detection of soluble products is achieved by coupling a unique chemical probe to the reducing end of oligosaccharides in a stable oxime linkage, while the use of (13)C-labeled monosaccharide standards (xylose and glucose) allows quantitation of the derivatized glycans. We apply this oxime-based nanostructure-initiator mass spectrometry (NIMS) method to characterize the functional diversity of GHs secreted by Clostridium thermocellum, a model cellulolytic organism. New reaction specificities are identified, and differences in rates and yields of individual enzymes are demonstrated in reactions with biomass substrates. Numerical analyses of time series data suggests that synergistic combinations of mono- and multifunctional GHs can decrease the complexity of enzymes needed for the hydrolysis of plant biomass during the production of biofuels.

Discovery of two novel ß-glucosidases from an Amazon soil metagenomic library.

An Amazon soil microbial community metagenomic fosmid library was functionally screened for ß-glucosidase activity. Contig analysis of positive clones revealed the presence of two ORFs encoding novel ß-glucosidases, AmBGL17 and AmBGL18, from the GH3 and GH1 families, respectively. Both AmBGL17 and AmBGL18 were functionally identified as ß-glucosidases. The enzymatic activity of AmBGL17 was further characterized. AmBGL17 was tested with different substrates and showed highest activity on pNPßG substrate with an optimum temperature of 45 °C and an optimum pH of 6. AmBGL17 showed a Vmax of 116 mM s(-1) and Km of 0.30 ± 0.017 mM. This is the first report of ß-glucosidases from an Amazon soil microbial community using a metagenomic approach.

Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates.

INTRODUCTION: Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. METHOD: We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. RESULTS: Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. CONCLUSION: Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

From soil to structure, a novel dimeric ß-glucosidase belonging to glycoside hydrolase family 3 isolated from compost using metagenomic analysis.

A recent metagenomic analysis sequenced a switchgrass-adapted compost community to identify enzymes from microorganisms that were specifically adapted to switchgrass under thermophilic conditions. These enzymes are being examined as part of the pretreatment process for the production of "second-generation" biofuels. Among the enzymes discovered was JMB19063, a novel three-domain ß-glucosidase that belongs to the GH3 (glycoside hydrolase 3) family. Here, we report the structure of JMB19063 in complex with glucose and the catalytic variant D261N crystallized in the presence of cellopentaose. JMB19063 is first structure of a dimeric member of the GH3 family, and we demonstrate that dimerization is required for catalytic activity. Arg-587 and Phe-598 from the C-terminal domain of the opposing monomer are shown to interact with bound ligands in the D261N structure. Enzyme assays confirmed that these residues are absolutely essential for full catalytic activity.

In vitro recombination of non-homologous genes can result in gene fusions that confer a switching phenotype to cells.

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 ß-lactamase (BLA) can result in fusion proteins in which ß-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to ß-lactam even though they do not encode allosteric enzymes. These 'phenotypic switch' genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.

Morphogen-defined patterning of Escherichia coli enabled by an externally tunable band-pass filter.

BACKGROUND: Gradients of morphogens pattern cell fate - a phenomenon that is especially important during development. A simple model system for studying how morphogens pattern cell behavior would overcome difficulties inherent in the study of natural morphogens in vivo. A synthetic biology approach to building such a system is attractive. RESULTS: Using an externally-tunable band-pass filter paradigm, we engineered Escherichia coli cells to function as a model system for the study of how multiple morphogens can pattern cell behavior. We demonstrate how our system exhibits behavior such as morphogen crosstalk and how the cells' growth and fluorescence can be patterned in a number of complex patterns. We extend our cell patterning from 2D cultures on the surface of plates to 3D cultures in soft agarose medium. CONCLUSION: Our system offers a convenient, well-defined model system for fundamental studies on how multiple morphogen gradients can affect cell fate and lead to pattern formation. Our design principles could be applied to eukaryotic cells to develop other models systems for studying development or for enabling the patterning of cells for applications such as tissue engineering and biomaterials.

An externally tunable bacterial band-pass filter.

The current paradigm for tuning synthetic biological systems is through re-engineering system components. Biological systems designed with the inherent ability to be tuned by external stimuli will be more versatile. We engineered Escherichia coli cells to behave as an externally tunable band-pass filter for enzyme activity and small molecules. The band's location can be positioned within a range of 4 orders of magnitude simply by the addition of compounds to the growth medium. Inclusion in the genetic network of an enzyme-substrate pair that functions as an attenuator is a generalizable strategy that enables this tunability. The genetic circuit enabled bacteria growth to be patterned in response to chemical gradients in nonintuitive ways and facilitated the isolation of engineered allosteric enzymes. The application of this strategy to other biological systems will increase their utility for biotechnological applications and their usefulness as a tool for gaining insight into nature's underlying design principles.

As easy as flipping a switch?

Proteins that behave as switches help to establish the complex molecular logic that is central to biological systems. Aspiring to be nature's equal, researchers have successfully created protein switches of their own design; in particular, numerous and varied zinc-triggered switches have been made. Recent studies in which such switches have been readily identified from combinatorial protein libraries support the notion that proteins are primed to show allosteric behavior and that newly created ligand-binding sites will often be functionally coupled to the original activity of the protein. If true, this notion suggests that switch engineering might be more tractable than previously thought, boding well for the basic science, sensing and biomedical applications for which protein switches hold much promise.