Ulvan is a complex sulfated polysaccharide biosynthesized by green seaweed and contains predominantly rhamnose, xylose, and uronic acid sugars. Ulvan-degrading enzymes have only recently been identified and added to the CAZy ( www.cazy.org ) database as family PL24, but neither their structure nor catalytic mechanism(s) are yet known. Several homologous, new ulvan lyases, have been discovered in Pseudoalteromonas sp. strain PLSV, Alteromonas LOR, and Nonlabens ulvanivorans, defining a new family PL25, with the lyase encoded by the gene PLSV_3936 being one of them. This enzyme cleaves the glycosidic bond between 3-sulfated rhamnose (R3S) and glucuronic acid (GlcA) or iduronic acid (IdoA) via a β-elimination mechanism. We report the crystal structure of PLSV_3936 and its complex with a tetrasaccharide substrate. PLSV_3936 folds into a seven-bladed β-propeller, with each blade consisting of four antiparallel β-strands. Sequence conservation analysis identified a highly conserved region lining at one end of a deep crevice on the protein surface. The putative active site was identified by mutagenesis and activity measurements. Crystal structure of the enzyme with a bound tetrasaccharide substrate confirmed the identity of base and acid residues and allowed determination of the catalytic mechanism and also the identification of residues neutralizing the uronic acid carboxylic group. The PLSV_3936 structure provides an example of a convergent evolution among polysaccharide lyases toward a common active site architecture embedded in distinct folds.

Ulvan is a complex sulfated polysaccharide present in the cell wall of green algae of the genus (Chlorophyta). The first ulvan-degrading polysaccharide lyases were identified several years ago, and more were discovered through genome sequencing of marine bacteria. Ulvan lyases are now grouped in three polysaccharide lyase (PL) families in the CAZy database, PL24, PL25, and PL28. The recently determined structures of the representative lyases from families PL24 and PL25 show that they adopt a seven-bladed β-propeller fold and utilize the His/Tyr catalytic mechanism. No structural information is yet available for PL28 ulvan lyases. NLR48 from belongs to PL28 together with its close paralog, NLR42. Biochemical studies of NLR42 have revealed that it can cleave ulvan next to both uronic acid epimers. We report the crystal structure of ulvan lyase NLR48 at 1.9-Å resolution. It has a β-jelly roll fold with an extended, deep, and positively charged substrate-binding cleft. Putative active-site residues were identified from the sequence conservation pattern, and their role was confirmed by site-directed mutagenesis. The structure of an inactive K162M mutant with a tetrasaccharide substrate showed the substrate occupying the "-" subsites. Comparison with lyases from other PL families with β-jelly roll folds supported assignment of the active site and explained its ability to degrade ulvan next to either epimer of uronic acid. NLR48 contains the His/Tyr catalytic machinery with Lys and Tyr playing the catalytic base/acid roles.

Ulvan is a major cell wall component of green algae of the genus Ulva and some marine bacteria encode enzymes that can degrade this polysaccharide. The first ulvan degrading lyases have been recently characterized and several putative ulvan lyases have been recombinantly expressed, confirmed as ulvan lyases and partially characterized. Two families of ulvan degrading lyases, PL24 and PL25, have recently been established. The PL24 lyase LOR_107 from the bacterial Alteromonadales sp. strain LOR degrades ulvan endolytically, cleaving the bond at the C4 of a glucuronic acid. However, the mechanism and LOR_107 structural features involved are unknown. We present here the crystal structure of LOR_107, representing the first PL24 family structure. We found that LOR_107 adopts a seven-bladed β-propeller fold with a deep canyon on one side of the protein. Comparative sequence analysis revealed a cluster of conserved residues within this canyon, and site-directed mutagenesis disclosed several residues essential for catalysis. We also found that LOR_107 uses the His/Tyr catalytic mechanism, common to several PL families. We captured a tetrasaccharide substrate in the structures of two inactive mutants, which indicated a two-step binding event, with the first substrate interaction near the top of the canyon coordinated by Arg-320, followed by sliding of the substrate into the canyon toward the active-site residues. Surprisingly, the LOR_107 structure was very similar to that of PL25 family PLSV_3936, despite only ~14% sequence identity between the two enzymes. On the basis of our structural and mutational analyses, we propose a catalytic mechanism for LOR_107 that differs from the typical His/Tyr mechanism.Ulvan is one of the two major cell wall components of marine green algae (genus Ulva and Enteromorpha). It is a complex sulfated polysaccharide composed mainly of 3-sulfated rhamnose (Rha3S), glucuronic acid (GlcA), iduronic acid (IdoA) and xylose (1). The common disaccharide repetitive units within the ulvan polysaccharides are [→4)-β-D-GlcA-(1→4)-α-LRha3S-(1→] called type A ulvanobiourinic-3-sulfate (A 3S ) and [→4)-α-L-IdoA-(1→4)-α-LRha3S(1→] called type B ulvanobiouronic-3-sulfate (B 3S ) (1) (Schema 1). The presence of iduronic acid and sulfated rhamnose differentiates ulvan from other polysaccharides of marine origin and displays similarity with mammalian glycosaminoglycans such as chondroitin sulfate and hyaluronic acid. This distinctive chemical feature makes ulvan an attractive candidate for various biomedical, nanobiotechnological and drug delivery applications (2-6).Polysaccharides containing uronic acid sugars can be degraded by enzymes utilizing a β-http://www.jbc.org/cgi/doi/10.1074/jbc.RA117. (PLs). They utilize a β-elimination mechanism to cleave the oxygenaglycone bond by abstracting the C5 proton, which results in the formation of an unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (ΔUA) at the non-reducing end of the oligosaccharide product (7). These enzymes are presently classified into 26 s...

Glycosaminoglycans (GAGs) are linear polysaccharides comprised of disaccharide repeat units, a hexuronic acid, glucuronic acid or iduronic acid, linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine. GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix, control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior and differentiation. Niche adapted bacteria express enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. PL have been classified into 24 sequence-related families. Comparison of 3D structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level, and identified occurrences of convergent evolution. We have characterized structurally and enzymatically heparinase III from Bacteroides thetaiotaomicron (BtHepIII; gene BT4657), which is classified within the PL12 family. BtHepIII is a 72.5 kDa protein. We present the X-ray structures of two crystal forms of BtHepIII at resolution 1.8 and 2.4 Å. BtHepIII contains two domains, the N-terminal α-helical domain forming a toroid and the C-terminal β-sheet domain. comparison with other GAG lyases, we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of BtHepIII toward sulfate-poor heparan sulfate substrate.

Ulvan is a complex sulfated polysaccharide biosynthesized by marine green algae and constitutes one of the two major polysaccharides of their cell wall. This water-soluble polysaccharide is composed predominantly of 3-sulfated rhamnose (R3S), glucuronic acid (GluA), iduronic acid (IdoA) and xylose. The physicochemical and biological properties of ulvan make it of interest for a variety of industrial applications. Bacteria cohabiting with the green algae contain enzymes able to degrade ulvan by a lytic β-elimination mechanism. Genes coding such lyases have been discovered in the genomes of several bacteria. Pseudoalteromonas sp. strain PLSV gene PLSV_3936 encodes an ulvan lyase that cleaves the glycosidic bond between 3-sulfated rhamnose (R3S) and glucuronic acid (GluA) or iduronic acid (IdoA). Another ulvan lyase, discovered in Alteromonadales and encoded by the gene LOR_107, degrades ulvan endolyticaly cleaving the bond between the rhamnose-3-sulfate and glucuronic acid. We have characterized biochemically these two lyases and determined their three-dimensional structures. They represent the first structures of lyases capable of degrading ulvan. In spite of only 17% sequence identity, these two enzymes share the same 7-bladed β propeller fold. The putative active site was identified from structure conservation and confirmed by mutagenesis and structures of these enzymes with bound tetrasaccharide substrates. The catalytic residues are histidine and tyrosine while the substrate acidic group is neutralized by an arginine. Metal ions were detected in both lyases but they play only structural roles and are not involved directly in the catalysis.