Breeanna R. Urbanowicz

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1A903V and CESA3T942I in Arabidopsis thaliana. Using 13C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1A903V and CESA3T942I displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1A903V and CESA3T942I have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein.

The hemicellulose 4-O-methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4-O-methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl-l-methionine to O-4 of α-d-glucopyranosyluronic acid residues that are linked to O-2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co2+ for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4-O-methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O-methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O-methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall.

Complex pectin metabolism by gut bacteria reveals novel catalytic functions

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.

Structural Organization and a Standardized Nomenclature for Plant Endo-1,4-β-Glucanases (Cellulases) of Glycosyl Hydrolase Family 9

Glycosyl, or glycoside, hydrolases (GHs) comprise a structurally diverse group of enzymes that hydrolyze glycosidic bonds between carbohydrates, or between carbohydrates and other noncarbohydrate moieties, and that collectively exhibit a wide range of substrate specificities. GH enzymes from across

A Tomato Endo-β-1,4-glucanase, SlCel9C1, Represents a Distinct Subclass with a New Family of Carbohydrate Binding Modules (CBM49)

A critical structural feature of many microbial endo-β-1,4-glucanases (EGases, or cellulases) is a carbohydrate binding module (CBM), which is required for effective crystalline cellulose degradation. However, CBMs are absent from plant EGases that have been biochemically characterized to date, and accordingly, plant EGases are not generally thought to have the capacity to degrade crystalline cellulose. We report the biochemical characterization of a tomato EGase, Solanum lycopersicum Cel8 (SlCel9C1), with a distinct C-terminal noncatalytic module that represents a previously uncharacterized family of CBMs. In vitro binding studies demonstrated that this module indeed binds to crystalline cellulose and can similarly bind as part of a recombinant chimeric fusion protein containing an EGase catalytic domain from the bacterium Thermobifida fusca. Site-directed mutagenesis studies show that tryptophans 559 and 573 play a role in crystalline cellulose binding. The SlCel9C1 CBM, which represents a new CBM family (CBM49), is a defining feature of a new structural subclass (Class C) of plant EGases, with members present throughout the plant kingdom. In addition, the SlCel9C1 catalytic domain was shown to hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and a variety of plant cell wall polysaccharides.

Topology of the Maize Mixed Linkage (1→3),(1→4)-β-D-Glucan Synthase at the Golgi Membrane

Mixed-linkage (1→3),(1→4)-β-d-glucan is a plant cell wall polysaccharide composed of cellotriosyl and cellotetraosyl units, with decreasingly smaller amounts of cellopentosyl, cellohexosyl, and higher cellodextrin units, each connected by single (1→3)-β-linkages. (1→3),(1→4)-β-Glucan is synthesized in vitro with isolated maize (Zea mays) Golgi membranes and UDP-[14C]d-glucose. The (1→3),(1→4)-β-glucan synthase is sensitive to proteinase K digestion, indicating that part of the catalytic domain is exposed to the cytoplasmic face of the Golgi membrane. The detergent {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid} (CHAPS) also lowers (1→3),(1→4)-β-glucan synthase activity. In each instance, the treatments selectively inhibit formation of the cellotriosyl units, whereas synthesis of the cellotetraosyl units is essentially unaffected. Synthesis of the cellotriosyl units is recovered when a CHAPS-soluble factor is permitted to associate with Golgi membranes at synthesis-enhancing CHAPS concentrations but lost if the CHAPS-soluble fraction is replaced by fresh CHAPS buffer. In contrast to other known Golgi-associated synthases, (1→3),(1→4)-β-glucan synthase behaves as a topologic equivalent of cellulose synthase, where the substrate UDP-glucose is consumed at the cytosolic side of the Golgi membrane, and the glucan product is extruded through the membrane into the lumen. We propose that a cellulose synthase-like core catalytic domain of the (1→3),(1→4)-β-glucan synthase synthesizes cellotetraosyl units and higher even-numbered oligomeric units and that a separate glycosyl transferase, sensitive to proteinase digestion and detergent extraction, associates with it to add the glucosyl residues that complete the cellotriosyl and higher odd-numbered units, and this association is necessary to drive polymer elongation.

The synthesis and origin of the pectic polysaccharide rhamnogalacturonan II - insights from nucleotide sugar formation and diversity

There is compelling evidence showing that the structurally complex pectic polysaccharide rhamnogalacturonan II (RG-II) exists in the primary cell wall as a borate cross-linked dimer and that this dimer is required for the assembly of a functional wall and for normal plant growth and development. The results of several studies have also established that RG-II structure and cross-linking is conserved in vascular plants and that RG-II likely appeared early in the evolution of land plants. Two features that distinguish RG-II from other plant polysaccharides are that RG-II is composed of 13 different glycoses linked to each other by up to 22 different glycosidic linkages and that RG-II is the only polysaccharide known to contain both apiose and aceric acid. Thus, one key event in land plant evolution was the emergence of genes encoding nucleotide sugar biosynthetic enzymes that generate the activated forms of apiose and aceric acid required for RG-II synthesis. Many of the genes involved in the generation of the nucleotide sugars used for RG-II synthesis have been functionally characterized. By contrast, only one glycosyltransferase involved in the assembly of RG-II has been identified. Here we provide an overview of the formation of the activated sugars required for RG-II synthesis and point to the possible cellular and metabolic processes that could be involved in assembling and controlling the formation of a borate cross-linked RG-II molecule. We discuss how nucleotide sugar synthesis is compartmentalized and how this may control the flux of precursors to facilitate and regulate the formation of RG-II.

Methods for structural characterization of the products of cellulose- and xyloglucan-hydrolyzing enzymes.

Structural characterization of oligosaccharide products generated by enzymatic hydrolysis of plant cell wall polysaccharides provides valuable information about the enzymes activity and substrate specificity. In this chapter, we describe some of the chemical, chromatographic, and spectroscopic methods that we routinely use to isolate and characterize oligosaccharides formed by enzymatic fragmentation of cellulose and xyloglucan. These include techniques to determine glycosyl residue and glycosyl linkage compositions by gas chromatography and mass spectrometry. We also illustrate the use of electrospray ionization with multistage mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy to perform detailed structural analysis of these oligosaccharides.

Loss of function of folylpolyglutamate synthetase 1 reduces lignin content and improves cell wall digestibility in Arabidopsis

BackgroundOne-carbon (C1) metabolism is important for synthesizing a range of biologically important compounds that are essential for life. In plants, the C1 pathway is crucial for the synthesis of a large number of secondary metabolites, including lignin. Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factors for C1 metabolic pathway enzymes. Given the link between the C1 and phenylpropanoid pathways, we evaluated whether folylpolyglutamate synthetase (FPGS), an enzyme that catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, can be a viable target for reducing cell wall recalcitrance in plants.ResultsConsistent with its role in lignocellulosic formation, FPGS1 was preferentially expressed in vascular tissues. Total lignin was low in fpgs1 plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 was mainly due to lower guaiacyl (G) lignin levels. Glycome profiling revealed subtle alterations in the cell walls of fpgs1. Further analyses of hemicellulosic polysaccharides by NMR showed that the degree of methylation of 4-O-methyl glucuronoxylan was reduced in the fpgs1 mutant. Microarray analysis and real-time qRT-PCR revealed that transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants. Consistent with the transcript changes of C1-related genes, a significant reduction in S-adenosyl-l-methionine content was detected in the fpgs1 mutant. The modified expression of the various methyltransferases and lignin-related genes indicate possible feedback regulation of C1 pathway-mediated lignin biosynthesis.ConclusionsOur observations provide genetic and biochemical support for the importance of folylpolyglutamates in the lignocellulosic pathway and reinforces previous observations that targeting a single FPGS isoform for down-regulation leads to reduced lignin in plants. Because fpgs1 mutants had no dramatic defects in above ground biomass, selective down-regulation of individual components of C1 metabolism is an approach that should be explored further for the improvement of lignocellulosic feedstocks.

Structural, mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water-mediated mechanism

The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.