The 24-chain core-shell nanostructure of wood cellulose microfibrils in seed plants

Wood cellulose microfibrils (CMFs) are the most abundant organic substance on earth, but their nanostructures are poorly understood. There are controversies regarding the glucan chain number (N) of CMFs during initial synthesis and whether they become fused afterwards. Here, we combined small-angle X-ray scattering (SAXS), solid-state nuclear magnetic resonance (ssNMR) and X-ray diffraction (XRD) analyses to resolve these controversies. We successfully developed SAXS measurement methods for the cross-section aspect ratio and area of the crystalline-ordered CMF core, which showed higher density than the semi-disordered shell. The 1:1 aspect ratio suggested that CMFs remain mostly segregated, not fused. The area measurement revealed the chain number in the core zone (Ncore). The ratio of ordered cellulose over total cellulose, termed Roc, was determined by ssNMR. Using the formula N = Ncore / Roc, we found that the majority of wood CMFs contain 24 chains, conserved between gymnosperm and angiosperm trees. The average wood CMF has a crystalline-ordered core of ~2.2 nm diameter and a semi-disordered shell of ~0.5 nm thickness. In naturally and artificially aged wood, we only observed CMF aggregation (contact without crystalline continuity) but not fusion (forming conjoined crystalline unit). This further argued against the existence of partially fused CMFs in new wood, overturning the recently proposed 18-chain fusion hypothesis. Our findings are important for advancing wood structural knowledge and more efficient utilization of wood resources in sustainable bio-economies.

Cellulose-Synthesizing Machinery in Bacteria

Cellulose is produced by all plants and a number of other organisms, including bacteria. The most representative cellulose-producing bacterial species is Gluconacetobacter xylinus (G. xylinus), an acetic acid bacterium. Cellulose produced by G. xylinus, commonly referred to as bacterial cellulose (BC), has exceptional physicochemical properties resulting in its use in a variety of applications. All cellulose-producing organisms that synthesize cellulose microfibrils have membrane-localized protein complexes (also called terminal complexes or TCs) that contain the enzyme cellulose synthase and other proteins. The bacterium G. xylinus is a prolific cellulose producer and a model organism for studies on cellulose biosynthesis. The widths of cellulose fibers produced by Gluconacetobacter are 50‒100 nm, suggesting that cellulose-synthesizing complexes are nanomachines spinning a nanofiber. At least four different proteins (BcsA, BcsB, BcsC, and BcsD) are included in TC from Gluconacetobacter, and the proposed function of each is as follows: BcsA, synthesis of a glucan chain through glycosyl transfer from UDP-glucose; BcsB, complexes with BcsA for cellulose synthase activity; BcsC, formation of a pore in the outer membrane through which a glucan chain is extruded; BcsD, regulates aggregation of glucan chains through four tunnel-like structures. In this review, we discuss structures and functions of these four and a few other proteins that have a role in cellulose biosynthesis in bacteria.

Structure of xyloglucan xylosyltransferase 1 reveals simple steric rules that define biological patterns of xyloglucan polymers

The plant cell wall is primarily a polysaccharide mesh of the most abundant biopolymers on earth. Although one of the richest sources of biorenewable materials, the biosynthesis of the plant polysaccharides is poorly understood. Structures of many essential plant glycosyltransferases are unknown and suitable substrates are often unavailable for in vitro analysis. The dearth of such information impedes the development of plants better suited for industrial applications. Presented here are structures of Arabidopsis xyloglucan xylosyltransferase 1 (XXT1) without ligands and in complexes with UDP and cellohexaose. XXT1 initiates side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. Structures here and the properties of mutant XXT1s are consistent with a SNi-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. The XXT1 dimer alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed by the acceptor binding cleft. Homology modeling of XXT2 and XXT5, the other two xylosyltransferases involved in xyloglucan biosynthesis, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. An assembly of the three XXTs can produce the xylosylation patterns of native xyloglucans, suggesting the involvement of an organized multienzyme complex in the xyloglucan biosynthesis.

Generation of Amylosucrase Variants That Terminate Catalysis of Acceptor Elongation at the Di- or Trisaccharide Stage

ABSTRACT An amylosucrase gene was subjected to high-rate segmental random mutagenesis, which was directed toward a segment encoding amino acids that influence the interaction with substrate molecules in subsites −1 to +3. A screen was used to identify enzyme variants with compromised glucan chain elongation. With an average mutation rate of about one mutation per targeted codon, a considerable fraction (82%) of the clones that retained catalytic activity were deficient in this trait. A detailed characterization of selected variants revealed that elongation terminated when chains reached lengths of only two or three glucose moieties. Sequencing showed that the amylosucrase derivatives had an average of no more than two amino acid substitutions and suggested that predominantly exchanges of Asp394 or Gly396 were crucial for the novel properties. Structural models of the variants indicated that steric interference between the amino acids introduced at these sites and the growing oligosaccharide chain are mainly responsible for the limitation of glucosyl transfers. The variants generated may serve as biocatalysts for limited addition of glucose moieties to acceptor molecules, using sucrose as a readily available donor substrate.

A Chemoenzymatic Route to Conjugatable β(1→3)-Glucan Oligosaccharides

3II-O-Allyl-α-laminaribiosyl fluoride was prepared as a key synthon for the enzymatic synthesis of β(1→3)-glucan oligosaccharides, catalyzed by a mutated β(1→3)-glucanase (E231G) from barley (Hordeum vulgare L.). A strategy was developed for enzymatic elongation of the β(1→3)-glucan chain from the reducing end, using a single glucoside acceptor. When β-glucoside phenyl disulfide was used as the acceptor, this methodology generated laminari-oligosaccharides conjugatable at both their reducing and non-reducing ends.

Mild Pfitzner—Moffat oxidation of the (1→3)-β-D-glucan from Saccharomyces cerevisiae

The structure of the cell wall glucan isolated from the industrial strain of Saccharomyces cerevisiae was characterized as to be composed of a linear (1→3)-β-D-glucan chain with single β-D-glucopyranosyl residues attached to every ninth backbone unit by (1→6)-glycosidic linkages. Mild oxidation of this β-D-glucan with a dimethyl sulfoxide—acetic anhydride reagent yielded an “oxidized” glucan with aldehyde groups introduced at C-6 and carbonyl oxygens located at C-2 and C-4 of the glucopyranosyl rings. The conversion of the oxidized glucan into the polyoxime was used to study the progress of oxidation and to establish the carbonyl groups distribution in this new reactive polysaccharide derived from baker’s yeast cell wall.