New Ulvan-Degrading Polysaccharide Lyase Family: Structure and

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New Ulvan-Degrading Polysaccharide Lyase Family: Structure and Catalytic Mechanism Suggests Convergent Evolution of Active Site Architecture ThirumalaiSelvi Ulaganathan,† Michal T. Boniecki,† Elizabeth Foran,‡ Vitaliy Buravenkov,‡ Naama Mizrachi,‡ Ehud Banin,‡ William Helbert,§ and Miroslaw Cygler*,†,∥ †

Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada Institute for Nanotechnology and Advanced Materials and Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat Gan, Israel § Recherches sur les Macromolécules Végétales, UPR-CNRS 5301, Université Joseph Fourier, and Institut de Chimie Moléculaire de Grenoble, ICMG, FR-CNRS 2607, Grenoble, France ∥ Department of Biochemistry, McGill University, Montreal, Quebec H3G 0B1, Canada ‡

S Supporting Information *

ABSTRACT: Ulvan is a complex sulfated polysaccharide biosynthesized by green seaweed and contains predominantly rhamnose, xylose, and uronic acid sugars. Ulvandegrading 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.

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catalytic mechanisms have been identified, indicating convergent evolution of these enzymes.2−4 Ulvan is a complex sulfated polysaccharide biosynthesized by marine green algae from genus Ulva and Enteromorpha and constitutes one of the two major polysaccharides of their cell wall.5 This water-soluble polysaccharide is composed predominantly of 3-sulfated rhamnose (R3S), glucuronic acid (GlcA), iduronic acid (IdoA), and xylose. The most common repetitive units within the ulvan polysaccharide are disaccharide units [→ 4)-β-D-GlcA-(1→4)-α-L-Rha3S-(1→] and [→4)-α-L-IdoA-(1→ 4)-α-L-Rha3S(1→], called type A ulvanobiourinic-3-sulfate (A3S) and type B ulvanobiouronic-3-sulfate (B3S), respectively.5 The presence of uronic acid in ulvan makes it susceptible to depolymerization by a lytic mechanism. It is to be noted that

large variety of polysaccharides exist in nature, and many enzymes have evolved for their synthesis, processing, and degradation (Carbohydrate-Active Enzymes (CAZy) Database, http://www.cazy.org/1). The predominant reaction mechanism for oligosaccharide degradation is hydrolysis, and more than 130 different glycosyl hydrolase (GH) families have been identified to date (CAZy Database). These enzymes cleave the bond between the anomeric carbon and the bridging oxygen of the glycosidic linkage. Various polysaccharides, such as glycosaminoglycans, pectin, and alginate, contain uronic acid residues (in which C6 is a carboxylate). These polysaccharides can be depolymerized by enzymes utilizing a lytic mechanisman elimination reaction in which the C5 proton (α- to the carboxylate) is abstracted and the sugar residue is expelled from C4 to give an α,β-unsaturated residue (Scheme 1). The number of polysaccharide lyase (PL) families is much smaller than that of glycosyl hydrolases, with 24 PL families identified so far (CAZy Database). Several different protein folds have been observed among PL families, but only two general © 2017 American Chemical Society

Received: February 10, 2017 Accepted: March 14, 2017 Published: March 14, 2017 1269

DOI: 10.1021/acschembio.7b00126 ACS Chem. Biol. 2017, 12, 1269−1280

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ACS Chemical Biology Scheme 1. Overall Reaction Catalyzed by Ulvan Lyase

identity to two other ulvan lyases coded by the Alteromonas LOR (LOR_29) and Nonlabens ulvanivorans (NLR_492) genes. Inspection of these ulvanolytic bacteria genomes revealed that these genes are located in the gene clusters of coregulated polysaccharide utilization loci (PUL). On the basis of sequence analysis, these lyases define a new polysaccharide lyase family, PL25 (CAZy, Dr. B. Henrissat, personal communication). In this report, we describe expression, purification, characterization, and crystallization of the PLSV_3936 ulvan lyase from the Pseudoalteromonas sp. PLSV strain. We have determined the crystal structure of this enzyme at 1.45 Å resolution and identified its fold as an all β-strand seven-bladed β-propeller. Furthermore, we have identified putative residues involved in catalysis and substrate binding and have determined the structure of the complex of PLSV_3936 with an ulvan tetrasaccharide substrate containing glucuronic acid. The key role of the identified residues in the enzyme’s activity was confirmed by site-directed mutagenesis and activity measurement of the mutants. Moreover, the enzymatic mechanism is proposed.

the physicochemical and biological properties of ulvan make it of interest for a variety of industrial applications in the agriculture, food, pharmaceutical, chemical, and biomaterials industries.6−8 Sulfated ulvan oligosaccharide has been reported as a synthetic elicitor for disease control in plants.9 In addition to medicine and agriculture, ulvan polysaccharide captured some attention in the environmental and bioenergy fields. Further identification and characterization of microbial enzymes for ulvan degradation will add the ability to utilize marine algae as a new source of useful industrial products.10 The first ulvan degrading activity was identified in Gramnegative bacteria isolated from mud.11 Identification of the unsaturated uronic acid4-deoxy-L-threo-hex-4-enopyranosiduronic acid (ΔUA)at the nonreducing end of oligosaccharide products characterized the enzyme as a lyase. Only recently have the first ulvan lyase genes been identified in the Gramnegative bacterium Nonlabens (Persicivirga) ulvanivorans strain PLR.12 One of these genes was cloned, expressed in Escherichia coli, and biochemically characterized as an endolytic ulvan lyase. The protein sequence of this novel lyase was unrelated to any other lyases and was the first representative of a new lyase family. Recent sequencing of the genome of N. ulvanivorans13 led to the identification of other putative ulvan lyases and also the first ulvan hydrolase, which was subsequently biochemically characterized.14 This enzyme has been classified as belonging to the GH105 CAZy family and displays unsaturated βglucuronyl activity, working together with ulvan lyases to fully degrade the ulvan polymer. Additionally, several genomes of ulvan-degrading bacteria were sequenced, including Pseudoalteromonas sp. strain PLSV,15 Alteromonas sp. strains LTR and LOR,16 and several more ulvan lyases were identified and some were partially characterized.17 In particular, the product of gene LOR_107 from Alteromonas sp. strain LOR was shown to have ulvan lyase activity and was recognized as the founding member of the polysaccharide lyase family PL24 with over 20 identified homologues. The Pseudoalteromonas PLSV (PLSV_3936) gene encodes a new ulvan lyase, which shows a high amino acid sequence

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RESULTS AND DISCUSSION Mining genomic databases revealed putative ulvan lyases that were unrelated either to the first described ulvan lyase of N. ulvanivorans or to the new ulvan lyase family PL24.17 They included PLSV_3936, PLSV_3902, and PLSV_3878 (Pseudoalteromonas PLSV) as well as LOR_29 (Alteromonas LOR sp.) and NLR_492 (Nonlabens ulvanivorans), which display ∼50−75% pairwise sequence identity. Altogether, these enzymes fulfill the criteria for establishing a new polysaccharide lyase family referred to as PL25 (B. Henrissat, personal communication). We have cloned, expressed, and purified ulvan lyase PLSV_3936 and succeeded in its crystallization with and without a tetrasaccharide substrate, which has led to the first structure determination of a polysaccharide lyase acting on ulvan. 1270


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ACS Chemical Biology Substrate Specificity. Degradation of ulvan by PLSV_3936 was monitored by chromatography, revealing its endoacting mode of action. PLSV_3936 incubated with ulvan led to the production of the same end products as obtained with LOR_29, attested to by gel permeation chromatography experiments (Figure 1). The end products, the disaccharide

length and are significantly longer. In particular, the fourth and fifth blades have long extensions on the topside of the propeller, and the connection between the first and second blade is quite long as well. As a consequence, the bottom of the cylinder is rather flat, with only a small depression in the center while a deep and elongated crevice is fomed at the top (Figure 2A). The walls of the crevice on two sides are made of the long extensions and are high; the wall of the crevice over the third blade is lower, forming a shallow neck as an entrance to the crevice (Figure 2B). A sulfate molecule is bound within this crevice, in the putative substrate binding site. Additionally, a metal ion is located in identical positions in both independent molecules. It is liganded by the side chains of His208, His264, His278, and Cys266 in a tetrahedral coordination. The metal has been identified as a Zn2+ ion based on its coordination geometry and its distance to the histidine NE2 or ND1 atoms of ∼2.05 Å and to an SG of cysteine of ∼2.27 Å. A fluorescence scan at the beamline confirmed the presence of zinc in the crystal. The Zn2+ ion is bound between the second and third blade of the propeller (Figure 2A,C). A phosphate ion is bound on the other side of His208, the zinc ligand, and forms additional hydrogen bonds with the side chains of His123, His143, Tyr188, and Arg204 (Figure 2A,C). Since ulvan is a negatively charged substrate, the binding pocket of PLSV_3936 was expected to have positively charged amino acids to facilitate the substrate. Indeed, the sides of the deep crevice on the top of the propeller are lined with basic and polar side chains while the bottom of the crevice has a more hydrophobic character. Highly Conserved Residues and Their Localization. The BLAST search18 for homologous protein sequences identified a large number of homologues. We have selected proteins with at least a 35% sequence identity over more than 65% of the PLSV_3936 length and aligned these sequences with CLUSTALX.19 The CONSURF program20 was employed to project the sequence conservation levels onto the threedimensional structure of PLSV_3936. Many conserved residues are in the interior of the protein not accessible to the solvent and are likely essential for maintaining protein fold. However, there is a cluster of surface-accessible conserved residues at one end of the crevice on the top of the protein (Figure 2D). This cluster includes fully conserved Asp121, His123, Lys125, His143, Tyr188, and Arg204 and highly conserved Gln66, Tyr246, and Arg282. Site Directed Mutagenesis of Putative Active Site Residues. On the basis of a visual inspection of the location of conserved residues within the cluster described above, we have selected the following residues for mutagenesis: His123, His143, Tyr188, Arg204, and His264. Our goal was to identify inactive mutants that we could utlize to capture the enzyme− substrate complex. In order to introduce minimal changes to the protein structure and to affect the shape of the substrate binding site as little as possible, we have replaced the side chains with their close steric analogs and constructed the subsequent mutants: H123N, H143N, Y188F, R204N, and H264N. With the exception of H143N, the remaining mutants showed no measurable activity against ulvan (Figure 3), indicating their essential role in catalysis or possibly in substrate binding. The significant remaining activity of the H143N mutant (65% activity of the wild type lyase) indicates that the contribution of His143 is less crucial. Structure of the PLSV_3936(H123N) Complexed with Ulvan Tetrasaccharide. We predicted His123 as a likely

Figure 1. Gel permeation chromatography of ulvan lyase end products. Top: Comparison of the end-products of LOR_29 and PLSV_3936 showed they are composed mainly of ΔUA-R3S and ΔUA-R3S-Xyl-R3S. Bottom: End products of LOR_107 composed notably of ΔUA-R3S-GlcA/IdoA-R3S were incubated with LOR_29 and PLSV_3936, confirming the cleavage of the bond next to both epimers of uronic acid.

ΔUA-Rha3S and tetrasaccharide ΔUA-Rha3S-Xyl-Rha3S, eluted at 88 and 84 min retention times, respectively (Figure 1). This differs from LOR_107 (PL24), whose end products contained two additional uronic tetrasaccharides, ΔUA-Rha3SGlcA/IdoA-Rha3S (81 min elution time). Therefore, PLSV_3936 can cleave the bond next to both GlcA and IdoA. This was confirmed by further degradation experiments where PLSV_3936 was able to completely convert the end products of LOR_107, containing the uronic tetrasaccharides, to the ΔUA-Rha3S disaccharide. This also indicates that PLSV_3936 can act on an oligosaccharide as short as a tetrasaccharide. Crystal Structure of PLSV_3936. The crystals of PLSV_3936 protein contain two molecules in the asymmetric unit. These two copies are virtually identical, and they superimpose with a root-mean-squares deviation (RMSD) of 0.21 Å for all Cα atoms. Each molecule adopts the fold of a seven-bladed β-propeller (Figure 2A) whereby each propeller consists of four antiparallel β-strands. Blades 1, 2, 3, 4, 5, and 6 are formed by consecutive residues, while blade 7 is formed from three C-terminal β-strands, with the fourth β-strand provided by the N-terminal residues 51−59 (Figure 2A). The loops joining β-strands are all rather short on one side of the cylinder formed by the blades (bottom) while the connecting loops on the opposite side of the cylinder (top) are of varied 1271


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Figure 2. Structural details of PLSV_3936. (A) Cartoon representation of one molecule of PLSV_3936. The molecule is rainbow colored from blue at the N-terminus to red at the C-terminus. The blades are numbered from 1 to 7. The Zn2+ ion is shown as a blue ball and the phosphate as orange and red balls. (B) The surface representation of the molecule, view on the top of the propeller. A deep crevice is located in the center with a neck leading to the outside (top of the figure). (C) The ligands of the Zn2+ and phosphate ions. (D) Level of conservation of surface exposed residues in PLSV_3936. The largest cluster of conserved surface residues is located at the upper end of the crevice (burgundy), next to the neck. The color scheme is shown below.

Figure 3. Activity of the wild type and mutants of PLSV_3936. Reaction conditions are 1 μg of enzyme in 20 mM Hepes 7.5 and 150 mM NaCl. Product formation was measured at 232 nm continuously for 15 min. Ulvan concentration was in each case 0.5 mg mL−1.

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Figure 4. Location and contact of the tetrasaccharide substrate. (A) The initial difference electron density contoured at the 3 s level around the substrate. The final refined position of the substrate is shown within the map. (B) The placement of the tetrasaccharide within the crevice. The surface of the protein is shown. The atoms of the substrate are shown as spheres (carbon, green; phosphate, yellow; oxygen, red). The R3S on the reducing end of the tetrasaccharide is at the top, located within the neck. (C) Direction of the oligosaccharide in the crevice. Protein surface is colored by atom type (carbon, white; oxygen, red; nitrogen, blue). Each sugar is depicted as a line connecting the C1 and C4 atoms (thick lines). The thinner lines depict connections between joined sugars and were drawn from C4 of the sugar on the reducing end to C1 of the sugar on the nonreducing end of the link. The first three sugars are extended along almost a straight line, and the last sugar follows the shape of the crevice and extends nearly perpendicularly the first three toward the surface.

Tyr246. There is one more hydrogen bond formed, between O3 of GlcA and ND2His208. The lyase makes several contacts with R3S in the −1 subsite. The R3S sulfate makes hydrogen bonds to Asn60, Asn123, Lys125, and Gln66 (to the latter through a bridging water molecule). The R3S O2 hydroxyl forms a hydrogen bond to Arg282, also through a bridging water molecule (Figure 5A), and the R3S C6 methyl group makes van der Waals contacts with methyl groups of Thr280 and Thr355 and with the Phe329 ring, contributing to the binding selectivity for this sugar. R3S in the +2 subsite makes predominantly van der Waals contacts with the protein, namely with the faces of His143, His158, and His208 and with Ala207, while the sulfate makes hydrogen bonds with His278 and Lys354 (weak). Finally, ΔUA extends toward the surface and makes only one hydrogen bond between its O2 and NH2Asn60. We next asked the question if the H123N mutation affected the position of the substrate. We modeled the His123 side chain in the lyase-substrate complex based on its conformation in the native crystal of PLSV_3936. In this model, the NE2His123 atom is ∼2.8 Å from the O4 oxygen bridging GlcA (reducing end) and R3S (nonreducing end) and pointing in the direction of a lone electron pair of the O4 atom. However, this NE2His123 is only 2.5 Å from the C5 atom (1.6 Å from the H−C5 proton). This short distance indicates that in the active enzyme the substrate must bind in a slightly shifted position, with the H− C5 moved away from His123 toward Tyr188. Tyr188 is proximal to the H−C5 proton, with its hydroxyl group extending toward this hydrogen and the HOTyr188 at ∼2.4 Å from H−C5 proton. This is a good geometry for proton abstraction. Moreover, His123 and Tyr188 are close to each other, with NE2His123 and OHTyr188 being in a hydrogen bonding distance. Proposed Catalytic Mechanism. The structure of the enzyme−substrate complex together with enzymatic data for

residue involved in catalysis. Crystals of the inactive H123N mutant were soaked with a tetrasaccharide mixture containing (nonreducing end) ΔUA-R3S-GlcA-R3S (reducing end) and ΔUA-R3S-IdoA-R3S. The resulting difference electron density map calculated at 1.6 Å resolution showed clear density for all four sugar residues and allowed identification of the uronic acid epimer as a glucuronic acid (Figure 4A). This suggested the preferred binding of the GlcA-containing tetrasaccharide over the one containing iduronic acid. The locations of the R3S sulfate groups were clear markers for placing the tetrasaccharide in the electron density and determining the directionality of the oligosaccharide. The tetrasaccharide assumes an L-shaped conformation within the crevice (Figure 4B). The R3S sugar on the reducing end lies within the entrance to the crevice, while the glucuronic acid and a second rhamnose-3-sulfate are located deep in the crevice. The last residue at the nonreducing end of the tetrasaccharide, ΔUA, extends toward the surface of the crevice, making a sharp turn relative to the direction of the first three sugars (Figure 4C). Three residues that were shown by mutagenesis as essential for activity, His123, Tyr188, and Arg204, make contact with the GlcA residue in the second position within the tetrasaccharide. Following the nomenclature introduced by Davies et al.,21 this GlcA sugar thus defines the +1 subsite, with the enzyme cleaving its C4−O4 bond. The remaining sugars of the tetrasaccharide occupy −2 (ΔUA), −1 (R3S), and +2 (R3S) subsites. The most contacts with the protein are made by the two middle sugars, GlcA (+1 subsite) and R3S (−1 subsite), and they are determinants of substrate recognition (Figure 5A). The lyase contacts GlcA on both sides of the sugar ring (Figure 5B). The acidic group of GlcA is positioned in exactly the same place as the phosphate ion in the native structure. It forms a salt bridge with Arg204 as well as hydrogen bonds with NE2His143 and OHTyr246 (Figure 5B). The arginine neutralizes the charge of the carboxylic group of GlcA with the aid of His143 and 1273


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Figure 5. Binding site of the ulvan tetrasaccharide substrate in the H123N mutant. (A) Stereo view of the residues in close proximity to the tetrasaccharide including the Zn2+ ion (gray sphere) and one bridging water molecule (red shere). Green dashed lines indicate hydrogen bonds. Red dashed lines indicate very close contacts. Blue dashed lines connect Zn2+ ion with liganding atoms. (B) Close-up of the central sugars GlcA and R3S and their contacts with neighboring side chains. Active side residue names are labeled with *. (C) The proposed reaction mechanism of PLSV_3936 with indicated roles of active site residues.

the mutants allowed us to deduce the catalytic cycle and firmly identify the residues participating in the catalysis. The mechanism, proposed originally by Gacesa22 and observed subsequently in all polysaccharide lyases, requires neutralization of the C5 acidic group on the uronic acid residue in the +1 position. In other polysaccharide lyases, this was accomplished either by a bound metal ion or an asparagine, aspartate, glutamine, glutamate, or histidine side chains.3,4 The neutralization of the C5 acidic group of the uronic acid in the +1 subsite is a prerequisite for making the C5 proton susceptible to an attack by a base. Our product analysis indicates that PLSV_3936 can break the bond next to either GlcA or IdoA. Since our structural data provide direct information about the binding of a substrate containing GlcA at the +1 position and

delineate residues involved in its degradation, we will discuss the breakdown specifically of this substrate. The acidic group of the substrate is neutralized by Arg204 with some help from His143 and Tyr246. The side chains in close proximity to H−C5 proton are Tyr188 and His123. According to our model, after slight repositioning of the substrate in the active site of the wild type enzyme (see above), the distance between the H−C5 proton and OHTyr188 or NE2His123 would be ∼2−2.5 Å, compatible with either of them acting as a base (Figure 5B). Nevertheless, the H−C5 bond would be inclined to the histidine ring, making proton transfer to His123 less likely. Tyr188, on the other hand, is pointing directly toward the H−C5, well poised for proton abstraction. His123 and Tyr188 are in close proximity, with NE2His123 and 1274


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Figure 6. Comparison of PLSV_3936 with other seven-bladed propellers. (A) Superposition of PLSV_3936 (green) and PL22 oligogalacturonan lyase, PDB code 3PE7 (yellow). Left view perpendicular to the barrel axis. Longer loops of PLSV_3936 on the top of the propeller are clearly visible. Right 90° rotation showing the view on the top of the propeller. (B) Comparison of the active site residues of PLSV_3936 (left) and putative catalytic residues of PL22 (right). The chosen orientations show the groups’ neutralizing charge of the acidic group of uronic acid (Arg204 in PLSV_3936 and Mn2+ in PL22) on the right and the catalytic residues on the left. The residues not only come from different blades but show different spatial arrangement. (C) The superposition of PLSV_3936 (green) and BACOVA_03493 (PDB code 4IRT, orange). The blades of the propellers superimpose well; the main differences are in the loops on the top of the propeller, which define the substrate binding site. The inset on the right shows the conservation of residues involved in catalysis in PLSV_3936.

OHTyr188 within 3.1 Å and forming a hydrogen bond in the wild type structure. When N123 in the H123N mutant-substrate complex is modeled back to His123, this distance would be even shorter, allowing for a proton transfer from Tyr188 to His123. This would result in a protonated form of His123 and deprotonated form of Tyr188, making Tyr188 ready to abstract the H−C5 proton. Since a proton has to be subsequently donated to the bridging oxygen, His123 appears to be best positioned for this action with the ring NE2 being ∼2.5 Å away from the bridging oxygen O4, which is already in the plane of the histidine ring. Although OHTyr188 is only ∼3.5 Å away from this bridging oxygen, the hydroxyl group direction is nearly perpendicular to the (R3S)C1−O4−C4(GlcA) plane and in a less favorable geometry for proton transfer to this O4. Importantly, since the position of the substrate in the wild type enzyme might be slightly different due to the fact that H123N replacement creates a small additional space for the substrate to move into, we cannot be absolutely certain about the precise roles of His123 and Tyr188 from the structure alone. Since the Y188F mutation inactivates the enzyme, we postulate that this side chain plays a major role in catalysis and is the most likely candidate for a base, while His123 would then transfer one of its two protons to the bridging oxygen. A concomitant breaking of the C4−O4 bond and the formation of a C4−C5 double bond would subsequently occur. It is not clear at this point if Tyr188 and His123 act in concert as a

proton relay or if there is a deprotonation at C5 followed by elimination. These could be either E2 or E1CB mechanisms, but we favor the latter since it is a syn elimination. The proposed mechanism is summarized in Figure 5C. Further experiments would be required to establish this firmly. The H264N mutation also inactivated the enzyme, although this side chain is not in contact with the substrate. The structure provides an answer for its role. His264 together with His208, His278, and Cys266 are ligands of the Zn2+ ion. Replacement of His264 by an asparagine likely results in the loss of Zn2+ binding. This metal plays a structural role, and its removal would destabilize the region near the active and substrate binding sites, which would inactivate the enzyme. This mutant precipitates readily during purification under the conditions where the wild type and other mutants are soluble, supporting the notion that some structural changes have taken place. The mechanism of breaking the bond next to IdoA is less clear at the present time. The findings that mutations of Arg204, His123, and Tyr188 completely inactivate the lyase suggest that these side chains are involved in catalysis of both uronic acid epimers. But the orientation and conformation of the +1 IdoA would have to be different from that of GlcA, and it is less clear which residues would be in the best position to act as the base and the acid. There are two other side chains in the vicinity of the +1 sugar, namely His208 and Tyr246, that 1275


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Figure 7. Similarity of the arrangement of the catalytic residues of PLSV_3936 (PL25) and PL15 exotype alginate lyase from Agrobacterium fabrum str. C58 (PL15, PDB code 3AFL) despite completely different folds exemplifies convergent evolution of these two families. (A) Stereo view (cartoon representation) of the superposition of PLSV_3936 (7-bladed β-propeller) and PL15 lyase ((α/α)5,6 incomplete toroid + antiparallel β-sandwich; the putative active site is located within the a (α/α)5,6 incomplete toroid domain) based on active site His, Tyr, and Arg residues. The active site residues are shown as sticks (center). (B) A close-up of the superimposed catalytic residues. The positions of the arginine side chain differ the most, likely due to a different orientation of the oligosaccharide substrates in the respective binding sites.

number of proteins with a similar seven-bladed β-propeller fold. This fold was seen frequently in enzymes as well as in protein domains that bind other proteins. However, closer inspection shows that a good fit is limited for most of them to a subset of blades. The best fit encompassing all blades is with a putative neuraminidase from Bacteroides ovatus, BACOVA_03493 (427 aa, PDB code 4IRT, Joint Center for Structural Genomics). The superposition of PLSV_3936 and 4IRT results in an RMSD of 1.64 Å for 267 Cα atoms (sPDBViewer26) and 2.8 Å for 366 Cα atoms (DALI24) with all blades showing a good overlap (Figure 6C). The amino acid sequence identity derived from the structural superposition is only 15.3%, indicating a distant evolutionary relationship (Figure S1). The BACOVA_03493 is annotated as a putative neuraminidase, and its substrate would also be an oligosaccharide chain. Therefore, we have carefully examined if any of the highly conserved PLSV_3936 amino acids in the surface cluster forming the active site are also present in BACOVA_03493. We found that the key PLSV_3936 residues are indeed conserved, namely His90 (His123 in PLSV_3936), His110 (His143), Tyr143 (Tyr188), Arg159 (Arg204), and Tyr197 (Tyr246). Many other residues conserved in PLSV_3936 and outside of this cluster were also present in BACOVA_03493 (Figure 6C). Despite a low sequence identity between these two proteins, we rationalize that their catalytic mechanisms are the same, and that BACOVA_03493 is a lyase with a so far unknown substrate rather than a neuraminidase, as annotated. Our conclusion is further supported by the analysis of the surrounding genes. The BACOVA_03493 gene, annotated as bovatus_03783 in CAZy, is located in a cluster of genes that include bovatus_03782 (GH43) and bovatus_03784 (GH105). The enzymes from the GH105 family are known to hydrolyze the disaccharide end products of lyases. We have obtained a sample of purified BACOVA_03493 from The Scripps Research Institute (TSRI) and tested it against ulvan polysaccharide. We detected no activity against this substrate,

could fulfill these roles. The latter side chain is located on the opposite side of the uronic acid ring to that of Tyr188 and would be directed toward the C5 hydrogen in the IdoA. Therefore, Tyr246 might play the role of a base during the cleavage next to IdoA. A detailed understanding of the mechanism for IdoA-containing ulvan will have to await determination of the structure of PLSV_3936 complexed with the ΔUA-R3S-IdoA-R3S substrate. Comparison with Other Polysaccharide Lyases and Seven-Bladed Propeller Proteins. The β-propeller fold is relatively rare among polysaccharide lyases and has been observed in only two polysaccharide lyase families: an eightbladed propeller in PL11, rhamnogalacturonan lyases, and a seven-bladed propeller in PL22, oligogalacturonan lyases.3 Comparison of the PL22 oligogalacturonate lyase from Yersinia enterocolitica23 (PDB code 3PE7) with PLSV_3936 shows that they have roughly the same radius for the barrel, but PLSV_3936 has longer interstrand loops, in particular those that encompass the substrate binding site (Figure 6A). The best structure-based alignment of PLSV_3936 with the PL22 lyase superimposes blade n of the former with blade n − 1 in the latter, and the corresponding sequence alignment shows only ∼5% sequence identity. There is no structure of PL22 lyase with a substrate, and only putative catalytic residues have been proposed. A Mn2+ ion is present in the substrate binding site of this lyase and could play the role of charge neutralizer. His242 was proposed as a base, while Arg217 and His211 have also been suggested to play a role in catalysis.23 These residues are located on the fourth and fifth blade, while the catalytically important residues in PLSV_3936 are in the second and third blades (Figure 6B). Despite the same seven-bladed propeller fold, the presumed catalytic residues cannot be superimposed, and the participation of Mn2+ in catalysis of PL22 lyase suggests a distant, if any, evolutionary relationship. A search for a similar fold through the Protein Data Bank using DALI24 and deconSTRUCT25 servers identified a large 1276



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indicating that it is not an ulvan lyase. Initial tests of this enzyme against heparin, chondroitin, hyaluronan, and xanthan showed no activity against these substrates. Further experiments using a broad range of polysaccharide substrates that are available at the CERMAV (Centre de Recherches Sur les Macromolécules Végétales) are in progress. Convergence of the Catalytic Site. The first notice of convergent evolution among polysaccharide lyases stemmed from the comparison of PL1 and PL10 lyase families.2 Subsequent analysis of PL families when more structural representatives were characterized showed that the histidinetyrosine combination acting as an acid−base pair in ulvan lyase is a common thread for many polysaccharide lyases displaying a variety of folds (Table 3 in ref 3). This is one of only two general catalytic mechanisms observed so far among PL enzymes and suitable for syn and/or anti β-elimination.3,4 Most polysaccharide lyases utilizing this mechanism deploy Asn, Asp, Glu, Gln, or His as a neutralizer of the C5-acidic group, to force its protonation. As mentioned above, the other seven bladed β-propeller family, PL22, evolved to utilize the metal assisted mechanism, with a Mn2+ ion being the neutralizer. Similarly, enzymes from the PL11 family, displaying an eight bladed β-propeller fold, also utilize the metal assisted catalytic mechanism. The ulvan lyase differs from the other two families and expands the range of PL folds that adopted the histidine−tyrosine mechanism of catalysis. There is however one PL family, PL15, where like in PLSV_3936 a positively charged residue, an arginine, acts as a likely neutralizer of the acidic group.27 Although, the fold of PL15 enzymes, (α/α)5,6 incomplete toroid + antiparallel βsandwich, and that of ulvan lyase (seven bladed β-propeller) are completely different from that of PLSV_3936 (Figure 7A). Their active site residues show a very similar spatial arrangement (Figure 7B). Some difference in the disposition of the arginine might be due to a different orientation of the oligosaccharide substrate in the respective binding sites of PL15 lyase and PLSV_3936. The overall similarity of the active site indicates a convergent evolution toward the same active site geometry in unrelated protein folds. Conclusions. We have determined the first three-dimensional structure of an ulvan lyase, which is a member of a new polysaccharide lyase family PL25 in the CAZy database. The lyase folds into a seven-bladed propeller with an extended crevice on one side of the propeller. Alignment of homologous sequences provided information on the level of conservation across the protein structure and identified a cluster of conserved residues located in the large crevice on the surface of the protein. Mutation of selected residues inactivated the enzyme and suggested their involvement in catalysis. The structure of the lyase-substrate complex confirmed the deep crevice as the substrate binding site and identified the catalytic residues, agreeing with the conclusions from mutagenesis. The structure of the lyase-substrate complex allowed us to assign specific roles for the active site residues. The negative charge of the acidic group on the glucuronic acid is neutralized by Arg204 with help from His143 and Tyr246. Tyr188 is the most likely candidate for the catalytic base and His123 is the most likely general acid. Both these residues have been found to fulfill such roles in other lyases3,4 and are playing the same roles in a lyase with a different fold.

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MATERIALS AND METHODS

Cloning and Purification of PLSV_3936 and Other Lyases. The gene encoding the ulvan lyase PLSV_3936 was cloned without a signal peptide into the expression vector (pET28a) with C terminal 6xHis tag and expressed in BL21-codon plus (DE3)-Ripl Escherichia coli. The expressed protein contains residues 34−489. An overnight inoculum of the expression strain was subcultured into 1 L of Terrific Broth medium (ThermoFisher, Canada) supplemented with kanamycin and chloramphenicol. Cells were allowed to grow at 37 °C until the absorbance at 600 nm reached 1.5; then temperature was reduced to 20 °C and protein expression was induced by 0.5 mM isopropyl 1thio-β-D-galactopyranoside (IPTG). After overnight growth at 20 °C, cells were harvested by centrifugation at 4500g for 20 min, resuspended in a lysis buffer (20 mM Tris at pH 8, 200 mM NaCl, 5% glycerol, and 5 mM Imidazole), and lysed using cell disruptor (Constant Systems Ltd., UK). The soluble fraction was collected by centrifuging at 39 000g for 40 min, and PLSV_3936 was purified by cobalt metal affinity chromatography. The fraction eluted by 40 mM imidazole was concentrated and loaded on a S200 size exclusion column (GE Life Sciences). The protein eluted as a single peak with the apparent molecular weight corresponding to a monomer. The protein was concentrated to 29 mg mL−1 for crystallization screening experiments. Selenomethionine labeled protein was produced by inhibiting the methionine biosynthesis pathway.28 Briefly, an overnight inoculum in 100 mL of LB media was grown at 37 °C. The next day, the cells were spun down, and the pellet was resuspended in M9 minimal media. The resuspended cells were used in a subculture of 1 L of minimal media supplemented with 50 μg of kanamycin and 50 μg of chloramphenicol. Cells continued to grow at 37 °C until OD reached ∼1 (5−6 h). Then, 100 mg of hydrophobic amino acids lysine, phenylalanine, and threonine; 50 mg of isoleucine, leucine, and valine; and 60 mg of Lselenomethionine were added. After 15 min, protein expression was induced by the addition of 0.5 mM IPTG, and cells continued to grow at 20 °C for 16 h. Next, the cells were spun down and the SeMetlabeled protein was purified as described for the native protein and concentrated to 30 mg mL−1 for crystallization. LOR_107 was cloned without a signal peptide, expressed, and purified as described previously.17 LOR_29 was cloned without a signal peptide into the expression vector (pET28) with the C terminal 6xHis tag. Enzymes were expressed in T7 Express Competent E. coli (NEB) containing the pRIL plasmid (Stratagene; 34 μg/mL Cm). Batch cultures were inoculated 1:50 (v/v) with transformed cells grown overnight and incubated for 2.5 h at 37 °C to reach an optical density of 0.6−0.8 at 595 nm. Protein expression was induced with 0.1 mM IPTG for 18 h at 16 °C. After centrifugation, the pellet was resuspended in 0.1 M Tris-HCl (Sigma) and 0.2 M NaCl at pH 7.5. Cell were lysed using PopCulture reagent (Novagen) followed by centrifugation to remove bacterial debris. The supernatant was loaded onto a nickel-Sepharose column charged with 100 mM NiSO4 (GE Healthcare). After washing, the bound proteins were eluted with a linear gradient of imidazole ranging from 20 to 500 mM. Proteincontaining fractions were verified by SDS polyacrylamide gel electrophoresis. Site Directed Mutagenesis. Single mutants of putative active site residues, H143N, H123N, R204N, Y188F, and H264N, were made using the Quickchange site-directed method (Agilent) following manufacturer instructions and using KOD polymerase (EMD Millipore, Etobicoke Ontario). The presence of designed mutations was confirmed by DNA sequencing. The mutants were expressed in BL21-codon plus (DE3)-Ripl. All mutants were purified following the same protocol as described above for the wild-type protein. Crystallization of PLSV_3936. Crystallization screening was carried on in a 96-well sitting drop format using commercial screens and the Gryphon crystallization robot (ArtRobbins Instruments, Sunnyvale, CA). Initial crystals appeared in 3 days under several conditions containing PEG3500 as a precipitant. The conditions were optimized using the hanging drop vapor diffusion method. The best crystals grew from 20% PEG3500, 0.2 M ammonium acetate, and 0.1 1277


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ethylene glycol molecules, and 1129 water molecules. The refinement converged at Rwork = 0.134 and Rfree = 0.143. The stereochemistry of the model was checked with Phenix software. The pertinent data collection and refinement data are shown in Table 1. The coordinates and structure factors have been deposited to the Protein Data Bank with accession numbers 5UAM for the native structure and 5UAS for enzyme-tetrasaccharide complex. Crystallization of the PLSV_3936(H123N) Mutant. Purified PLSV_3936(H123N) was concentrated to approximately to 29 mg mL−1 and crystallized in the native conditions. These conditions were optimized, and the best crystals grew from 15% PEG3500, 0.2 M ammonium acetate, and 0.1 M Bis-Tris, at pH 5. Crystals of PLSV_3936(H123N) with Ulvan Tetrasaccharide Substrate. Crystals of PLSV_3936(H123N) were soaked for ∼5 h in solution containing two different tetrasaccharide substrates. The tetrasaccharides were obtained by enzymatic digestion of ulvan polysaccharide with LOR_107 ulvan lyase (PL24) from Alteromonas LOR sp. (Kopel et al.) and separated by HPLC chromatography. Tetrasaccharide I (DP4−1) contains a mixture of ΔUA-Rha3S-GlcARha3Sα/β and ΔUA-Rha3S-IdoA-Rha3Sα/β with some traces of hexasaccharides. Tetrasaccharide II (DP4−2) is a pure sample of ΔUA-Rha3S-Xyl-Rha3Sα/β. After soaking in tetrasaccharide solution, the crystals were briefly washed in the cryoprotectant solution containing 30% ethylene glycol in the mother liquor and flash frozen in liquid nitrogen. Diffraction data were collected at the 08B1 beamline at the CLS and processed with XDS. These crystals diffracted to 1.6 Å resolution and were isomorphous to the native crystals. They belonged to the space group C2 with the unit cell dimensions a = 173.6, b = 71.8, and c = 109.3 Å and β = 122.5° and two molecules in the asymmetric unit. A strong positive difference electron density corresponding to the bound substrate was observed in a crystal soaked in DP4−1, but no such density was found in the electron density map for the crystal soaked in DP-2. The difference electron density for that soaked in DP4−1 showed clearly the presence of a tetrasaccharide with glucuronic acid in the +1 position in both lyase molecules in the asymmetric unit. The final model contains residues 47−486 in chain A, 48−486 in chain B, two Zn2+ ions, two DP4 tetrasaccharides, five ethylene glycol molecules, and 11 230 waters. The refinement was performed with Phenix software and converged at Rwork = 0.182 and Rfree = 0.212 (Table 1). Activity Measurement of PLSV_3936 and Mutants on Ulvan Substrate. The enzymatic activity of PLSV_3936 and its mutants as well as LOR_107 and LOR_29 was measured using ulvan as a substrate. Ulvan was obtained from CEVA (Pleubian, France). The cleavage of a rhamnose-uronic acid bond leads to the appearance of an unsaturated C4C5 double bond within the uronic acid hexose ring, which modified the absorption spectrum of the ring with an absorption maximum at 232 nm. To follow the reaction, 0.5 mg mL−1 of ulvan polysaccharide was dissolved in 50 μL of a buffer containing 20 mM Hepes at pH 7.5 and 150 mM NaCl, and 1 μg of the purified enzyme was added directly to the assay solution. Absorption at 232 nm was measured continuously for 30 min. The absorbance of the substrate alone over the same period of time was used as a control. Where indicated, sequential enzymatic treatments were also carried out. Notably, ulvan was first treated with LOR_107 followed by treatment with PLSV_3926 and LOR_29. Substrate Specificity. To examine the difference in specificity between LOR_107, LOR_29, and PLSV393, ulvan degradation products of each enzyme as well as combinations were also monitored by analytical gel permeation chromatography. Samples were injected on a Superdex 200 (10/300 GL, GE Healthcare) and a peptide HR (10/300 GL, GE Healthcare) column coupled in series. Elution was conducted with an Ultimate 3000 HPLC system (ThermoScientific, Dionex) operating at 0.5 mL min−1 with 50 mM (NH4)2CO3 (pH 8) as the eluent. Detection of products was achieved with a RSLC UV detector (ThermoScientific, Dionex) operating at 235 nm, along with an IOTA 2 refractive index detector (Precision Instrument).

M Bis-Tris, at pH 5.5. For data collection, the crystals were briefly washed in a cryoprotectant solution (70% mother liquor and 30% ethylene glycol) and flash cooled in liquid nitrogen. Diffraction data were collected on the 08B1 beamline at the Canadian Light Source synchrotron (CLS)29 and processed with XDS. These crystals diffracted to 1.45 Å resolution and belonged to the space group C2, with unit cell dimensions a = 174.3, b = 72.1, and c = 109.5 Å and β = 122.5°. There are two molecules in the asymmetric unit. The best crystals of SeMet-labeled protein were grown from 20% PEG3500, 0.2 M ammonium acetate, and 0.1 M 4-morpholineethanesulfonic acid (MES), at pH 6.5. These crystals were briefly transferred to the mother liquor containing an additional 30% ethylene glycol and flash cooled in liquid nitrogen. Diffraction data to 1.6 Å resolution were collected on the 08B1 beamline at the CLS and processed using XDS. The crystals belong to the space group C2 with unit cell dimensions a = 174.2, b = 71.7, and c = 109.4 Å and β = 122.6°. Data collection statistics are summarized in Table 1.

Table 1. Statistics of Data Collection and Refinementa crystal form space group a, b, c (Å)

native PLSV_3936 C2 174.3, 72.1, 109.5 122.5 0.9791 49.01−1.45 763554 194580 96.2 (83.2)

β (deg) wavelength (Å) resolution (Å) observed hkl # unique hkl completeness (%) redundancy 3.7 Rsym 0.044 (0.713) CC1/2 99.9 (82.2) I/(σI) 19.15 (2.74) Rwork 0.134 Rfree 0.143 Wilson B (Å2) 16.5 ⟨B-factor⟩ (Å2; # atoms) protein 21.2 (7012) ligand 36.3 (58) solvent 34.5 (1128) Ramachandran plot favored (%) 96.8 allowed (%) 3.1 clash score 2.1 RMS deviation bonds (Å) 0.007 angles (deg) 1.00 PDB code 5UAM a b

SeMet complex PLSV_3936 PLSV_3936 C2 C2 173.6, 71.8, 109.1 173.6, 71.8, 109.3 122.4 0.9788 47.8−2.0 574743 145561b 98 (95.2)

122.4 0.9871 46.3−1.6 1094771 143664 96.0 (90.0)

3.9 11.1 (76.1) 99.7 (69.0) 11.23 (2.11)

7.6 0.097 (1.346) 99.9 (65.5) 13.75(1.5) 0.182 0.212 18.0 24.0 (6956) 28.2 (130) 36.4 (1128) 96.2 3.8 5.7 0.009 0.96 5UAS

Values in parentheses correspond to the highest resolution shell. F(+) and F(−) were counted as separate reflections.

Structure Determination and Refinement. The structure of PLSV_3936 was solved with the SeMet data using an AUTOSOL routine within the Phenix software.30 There are two molecules in the asymmetric unit; each contains 10 methionines. A total of 23 anomalous scatterers were found, and the initial model contained 787 out of 978 residues. This model was refined against a 1.45 Å resolution native data set using the Phenix software interspaced with manual rebuilding and solvent placement using COOT.31 Two strong peaks were observed in the mFo-DFc difference electron density map, one in each molecule, in identical positions. On the basis of the coordination and distances to the liganding atoms, these peaks were deemed to be Zn2+ ions. The final model contains residues 47 to 486 in chain A, 48 to 487 in chain B, two Zn2+ ions, one PO4 ion, one glycerol, nine 1278


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through the jasmonic acid signaling pathway. J. Biomed. Biotechnol. 2010, 525291. (10) Hehemann, J.-H., Boraston, A. B., and Czjzek, M. (2014) A sweet new wave: structures and mechanisms of enzymes that digest polysaccharides from marine algae. Curr. Opin. Struct. Biol. 28, 77−86. (11) Lahaye, M., Brunel, M., and Bonnin, E. (1997) Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp, (Ulvales, Chlorophyta). Carbohydr. Res. 304, 325−33. (12) Nyvall Collen, P., Sassi, J. F., Rogniaux, H., Marfaing, H., and Helbert, W. (2011) Ulvan lyases isolated from the Flavobacteria persicivirga ulvanivorans are the first members of a new polysaccharide lyase family. J. Biol. Chem. 286, 42063−71. (13) Kopel, M., Helbert, W., Henrissat, B., Doniger, T., and Banin, E. (2014) Draft Genome Sequence of Nonlabens ulvanivorans, an UlvanDegrading Bacterium. Genome announcements 2 (4), e00793-14. (14) Collen, P. N., Jeudy, A., Sassi, J. F., Groisillier, A., Czjzek, M., Coutinho, P. M., and Helbert, W. (2014) A novel unsaturated betaglucuronyl hydrolase involved in ulvan degradation unveils the versatility of stereochemistry requirements in family GH105. J. Biol. Chem. 289, 6199−211. (15) Kopel, M., Helbert, W., Henrissat, B., Doniger, T., and Banin, E. (2014) Draft Genome Sequence of Pseudoalteromonas sp. Strain PLSV, an Ulvan-Degrading Bacterium. Genome announcements 2 (6), e0125714. (16) Kopel, M., Helbert, W., Henrissat, B., Doniger, T., and Banin, E. (2014) Draft Genome Sequences of Two Ulvan-Degrading Isolates, Strains LTR and LOR, That Belong to the Alteromonas Genus. Genome announcements 2 (5), e01081-14. (17) Kopel, M., Helbert, W., Belnik, Y., Buravenkov, V., Herman, A., and Banin, E. (2016) New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order. J. Biol. Chem. 291, 5871− 5878. (18) Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389−3402. (19) Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., and Thompson, J. D. (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497−3500. (20) Landau, M., Mayrose, I., Rosenberg, Y., Glaser, F., Martz, E., Pupko, T., and Ben-Tal, N. (2005) ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299−302. (21) Davies, G. J., Wilson, K. S., and Henrissat, B. (1997) Nomenclature for sugar-binding subsites in glycosyl hydrolases. Biochem. J. 321, 557−9. (22) Gacesa, P. (1987) Alginate-modifying enzymes. A proposed unified mechanism of action for the lyases and epimerases. FEBS Lett. 212, 199−202. (23) Abbott, D. W., Gilbert, H. J., and Boraston, A. B. (2010) The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination. J. Biol. Chem. 285, 39029−39038. (24) Holm, L., and Rosenström, P. (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545−W549. (25) Zhang, Z. H., Bharatham, K., Sherman, W. A., and Mihalek, I. (2010) deconSTRUCT: general purpose protein database search on the substructure level. Nucleic Acids Res. 38, W590−W594. (26) Guex, N., and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714−2723. (27) Ochiai, A., Yamasaki, M., Mikami, B., Hashimoto, W., and Murata, K. (2010) Crystal structure of exotype alginate lyase Atu3025 from agrobacterium tumefaciens. J. Biol. Chem. 285, 24519−24528. (28) Walden, H. (2010) Selenium incorporation using recombinant techniques. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 352−7.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00126. Figure S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +1 306-966-4361. E-mail: [email protected]. ORCID

Miroslaw Cygler: 0000-0003-4579-1881 Funding

The financial support was provided by a grant from the Natural Science and Engineering Research Council of Canada (MC). E.B. would like to acknowledge Michael Abeles for his support and the Israel Ministry of Science, Technology and Space Infrastructure grant. W.H. acknowledges the French National Association for Research (ANR CE05-2014, GreenAlgohol). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Dr. D. R. Palmer for helpful discussions and to acknowledge the Protein Characterization and Crystallization Facility, College of Medicine, University of Saskatchewan for access to the crystallization robot. Research described in this paper was performed using beamline 08B1 at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research.

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REFERENCES

(1) Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., and Henrissat, B. (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490−D495. (2) Charnock, S. J., Brown, I. E., Turkenburg, J. P., Black, G. W., and Davies, G. J. (2002) Convergent evolution sheds light on the anti-beta -elimination mechanism common to family 1 and 10 polysaccharide lyases. Proc. Natl. Acad. Sci. U. S. A. 99, 12067−12072. (3) Garron, M. L., and Cygler, M. (2010) Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology 20, 1547−73. (4) Garron, M. L., and Cygler, M. (2014) Uronic polysaccharide degrading enzymes. Curr. Opin. Struct. Biol. 28, 87−95. (5) Lahaye, M., and Robic, A. (2007) Structure and Functional Properties of Ulvan, a Polysaccharide from Green Seaweeds. Biomacromolecules 8, 1765−1774. (6) Cunha, L., and Grenha, A. (2016) Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications. Mar. Drugs 14, 42. (7) Morelli, A., Betti, M., Puppi, D., and Chiellini, F. (2016) Design, preparation and characterization of ulvan based thermosensitive hydrogels. Carbohydr. Polym. 136, 1108−1117. (8) Manivasagan, P., and Oh, J. (2016) Marine polysaccharide-based nanomaterials as a novel source of nanobiotechnological applications. Int. J. Biol. Macromol. 82, 315−27. (9) Jaulneau, V., Lafitte, C., Jacquet, C., Fournier, S., Salamagne, S., Briand, X., Esquerre-Tugaye, M. T., and Dumas, B. (2010) Ulvan, a sulfated polysaccharide from green algae, activates plant immunity 1279


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New Ulvan-Degrading Polysaccharide Lyase Family: Structure and

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