Phenylpropanoid metabolism yields a mixture of monolignols that undergo chaotic, non-enzymatic reactions such as free radical polymerization and spontaneous selfassembly in order to form the polyphenolic lignin which is a barrier to cost-effective lignocellulosic biofuels. Post-synthesis lignin integration into the plant cell wall is unclear, including how the hydrophobic lignin incorporates into the wall in an initially hydrophilic milieu. Self-assembly, self-organization and aggregation give rise to a complex, 3D network of lignin that displays randomly branched topology and fractal properties. Attempts at isolating lignin, analogous to archaeology, are instantly destructive and nonrepresentative of in planta. Lack of plant ligninases or enzymes that hydrolyze specific bonds in lignin-carbohydrate complexes (LCCs) also frustrate a better grasp of lignin. Supramolecular self-assembly, nano-mechanical properties of lignin-lignin, ligninpolysaccharide interactions and association-dissociation kinetics affect biomass deconstruction and thereby cost-effective biofuels production. OPEN ACCESSMolecules 2010, 15 8642
Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses. We utilized transposon insertional mutagenesis to identify the P. multocida HA synthase, the enzyme that polymerizes HA. A DNA fragment from a wild-type genomic library could direct HA production in vivo in Escherichia coli, a bacterium that normally does not produce HA. Analysis of truncated plasmids derived from the original clone indicated that an open reading frame encoding a 972-residue protein was responsible for HA polymerization. This identification was confirmed by expression cloning in E. coli; we observed HA capsule formation in vivo and detected activity in membrane preparations in vitro. The polypeptide size was verified by photoaffinity labeling of the native P. multocida HA synthase with azido-UDP sugar analogs. Overall, the P. multocida sequence is not very similar to the other known HA synthases from streptococci, PBCV-1 virus, or vertebrates. Instead, a portion of the central region of the new enzyme is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular polysaccharides or lipopolysaccharides. In summary, we have discovered a unique HA synthase that differs in sequence and predicted topology from the other known enzymes.
We demonstrate in this report that the Xenopus DG42 gene product made in the yeast Saccharomyces cerevisiae can synthesize authentic high molecular weight hyaluronan (hyaluronic acid; HA) in vitro. Saccharomyces are eukaryotes that do not naturally produce HA or any other molecules known to contain glucuronic acid. Therefore bakers' yeast is a good model system to determine the enzymatic activity of the DG42 protein, which is the topic of recent debate. Membrane extracts prepared from cells expressing DG42 encoded on a plasmid incorporated [14C]glucuronic acid and N-[3H]acetylglucosamine from exogenously supplied UDP-sugar nucleotides into a high molecular mass (10(6) to 10(7) Da) polymer in the presence of magnesium ions. Both sugar precursors were simultaneously required for elongation. Control extracts prepared from cells with the vector plasmid alone or the DG42 cDNA in the antisense orientation did not display this activity. The double-labeled polysaccharide product synthesized in vitro was deemed to be HA by enzymatic analyses; specific HA lyase could degrade the polymer, but it was unaffected by protease or chitinase treatments. The fragments generated by HA lyase were identical to fragments derived from authentic vertebrate HA as compared by high performance liquid chromatography. We conclude that DG42 is a membrane-associated HA synthase capable of transferring both glucuronic acid and N-acetylglucosamine groups.
We have characterized the hyaluronan (HA) synthase activity of the Xenopus DG42 gene product in vitro. The recombinant enzyme produced in yeast does not possess a nascent HA chain and, therefore, is an ideal model system for kinetic studies of the synthase's glycosyltransferase activity. The enzymatic rate was optimal from pH 7.6 to 8.1. Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a large molecular weight polymer. UDP-glucose or the galactose epimers of the normal substrates did not substitute. The Michaelis constant, K m , of recombinant DG42 in membranes was 60 ؎ 20 and 235 ؎ 40 M for UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to values obtained previously from membranes derived from vertebrate cells. The apparent energy of activation for HA elongation is about 15 kilocalories/ mol. DG42 polymerizes HA at average rates of about 80 to 110 monosaccharides/s in vitro. The resulting HA polysaccharide possessed molecular weights spanning 2 ؋ 10 6 -10 7 Da, corresponding to about 10 4 sugar residues. This is the first report characterizing a defined eukaryotic enzyme that can produce a glycosaminoglycan. Glycosaminoglycans (GAG),1 linear polysaccharides based on a repeating disaccharide that usually consists of an amino sugar and a negatively charged sugar, are essential constituents of higher animals. Hyaluronan (HA), heparin, and chondroitan, dermatan, and keratan sulfates are members of this class of carbohydrates. HA (34) (13) is a prominent GAG that plays roles as a structural element and a recognition molecule in vertebrates (1). The enzymes that catalyze the production of HA, the HA synthases, were the first glycosyltransferases capable of forming the disaccharide repeat of a GAG to be cloned and described at the molecular level. The initial HA synthase to be identified was HasA of Streptococcus pyogenes which is the enzyme responsible for the formation of an extracellular capsule of HA in this human bacterial pathogen (2, 3). The HasA protein is strongly associated with the phospholipid membrane and is predicted to possess 4 or 5 membrane-spanning segments (3, 4). The enzyme utilizes UDP-GlcA and UDP-GlcNAc precursors found in the cytosol and extrudes the growing HA chain out of the cell during polymerization. HasA, a single protein, transfers both GlcA and GlcNAc residues to HA based on genetic and biochemical evidence (3,5).A Xenopus laevis (African clawed frog) protein, DG42 (for differentially expressed in gastrulation), with a previously unknown function (6) was found to be quite similar at the amino acid sequence level to the bacterial HasA enzyme (3, 4) as well as fungal chitin synthases (7). These observations led to the hypothesis that this vertebrate protein was also a HA synthase (4, 7). DG42 contains predicted transmembrane segments clustered at both the amino and carboxyl termini; this positioning is similar to that of the membrane-associated regions found in HasA (4). DG42 was s...
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