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ScienceDirect Uronic polysaccharide degrading enzymes Marie-Line Garron1,2 and Miroslaw Cygler3 In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the b-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide. Addresses 1 Aix-Marseille University, AFMB UMR7257, 163 Avenue de Luminy, 13288 Marseille, France 2 CNRS, AFMB UMR7257, 163 Avenue de Luminy, 13288 Marseille, France 3 Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada Corresponding author: Cygler, Miroslaw ([email protected])

Current Opinion in Structural Biology 2014, 28:87–95 This review comes from a themed issue on Carbohydrate-protein interactions and glycosylation Edited by Harry J Gilbert and Harry Brumer For a complete overview see the Issue and the Editorial Available online 25th August 2014 http://dx.doi.org/10.1016/j.sbi.2014.07.012 0959-440X/# 2014 Elsevier Ltd. All rights reserved.

Introduction Alongside nucleic acids, lipids and proteins, carbohydrates are the essential building blocks of living organisms. They are present in all three kingdoms of life and the chemical variety of their monosaccharides by far exceeds that of the standard amino acids, notwithstanding www.sciencedirect.com

the multitude of ways they are joined into oligo-saccharide or polysaccharide chains [1]. Glycans are involved in a wide range of biological activities, such as cell and tissue architecture, immunity, metabolism and pathogenesis. The structural variety of the oligosaccharides derives from the diversity of enzymes dedicated by various organisms to their synthesis and degradation [1,2]. Significant efforts led to the identification of Carbohydrate Active enZymes (CAZymes) in all sequenced genomes and their classification into families based on sequence similarities (www.cazy.org) [3,4,5]. Two kinds of enzymes are involved in polysaccharide degradation that break the O-linkage (C1-O-Cn) between two sugars, namely glycoside hydrolases (GHs) and polysaccharide lyases (PLs). GHs utilize a water molecule to break the anomeric C1–O bond (Figure 1) and are presently classified into 133 families and form the most abundant group of CAZymes. PLs are specific to the polysaccharides containing uronic acid and break the O–C4 bond to the uronic acid employing a b-elimination mechanism, which yields a 4,5-unsaturated sugar at the new non-reducing end of the product (Figure 1a). There are presently 23 PL families within the CAZy database, significantly fewer than the GH families, likely reflecting the smaller range of substrates. Two general enzymatic mechanisms were identified in PLs: the metal-assisted mechanism predominant in pectin degradation, and the histidine/tyrosine mechanism used by enzymes families involved in syn and/or anti b-elimination (Figure 1b) [6]. Previously, we discussed the relationship between the structural fold, substrate specificity and catalytic mechanism of the 21 PL families identified at that time [6]. Known PL structures adopted only six folds, with two families having no structural representatives. There is now at least one structural model for each of the 23 PL families. These new structures provided insight into the protein fold for family PL12 (heparinase III), PL17 (alginate lyases), PL22 (oligo-galacturonate lyase) and PL23 (viral chondroitin lyases). Moreover, the enzymatic mechanisms of PL4 and PL22 are now partially elucidated. Here we review the recent structural and functional data on PL enzymes and compare them with uronic polysaccharide hydrolases to find possible evolutionary relationships between GHs and PLs. Finally, we explore the new lyase activities identified among the ‘non classified’ lyases in CAZy database.

The subfamily classification of PLs A more comprehensive sequence analysis, which incorporated maximum likelihood phylogenetic analysis was Current Opinion in Structural Biology 2014, 28:87–95

88 Carbohydrate–protein interactions and glycosylation

Figure 1

(a)

O

(b)

– –

O2C

O HO

O2C

O

– O2C R O HO

O

O OH

OR H OH

HO

OH OR

H2O O



O HO

O2C

O H OH

O2C

R’ ROH

O

O

OH

O2C

HO HO

H OH OR

R O CO – 2 O R’ HO O H OH B:

O

H OH OR



anti ROH

anti

O HO

OH O

B:

B:





syn

O

R’

2C

ROH

syn

O HO

O –O

R O H

O2C

OH O

R’

OH O

R’

:B O

HO ROH

:B

H R O HO

–O

2C

OH O

R’

Current Opinion in Structural Biology

Action of polysaccharide lyases and glycoside hydrolyses. (a) The hydrolase and lyase catalyzed breakdown of hexose-uronate disaccharide. Cleavage resulting via elimination severs the C4–O bond of the glucuronyl residue and introduces a double bond into the +1 ring. Cleavage resulting from glycoside hydrolysis severs the C1–O bond of the adjacent (1) sugar residue; an inverting glycosidase reaction is shown; R = H or another sugar residue. (b) Elimination from D-glucoronide and L-iduronide residues proceeds via syn and anti-pathways, respectively, R = another sugar residue, R0 = H or another sugar residue.

applied to the sequences of PLs and led to the subdivision of the families into 61 subfamilies (Table S1). This new hierarchical classification demonstrated better correlation between the subfamily and the substrate specificity; 90% of the subfamilies for which functional data are now available appear to be mono-specific [5,7]. In this extended classification many PL subfamilies have no functionally or structurally characterized members and it is expected that novel specificities will be discovered and correlated with their structures.

Modularity and cooperativity of PLs As with other CAZymes, PLs are modular enzymes frequently associated with Carbohydrate Binding Modules (CBMs). CBMs are small domains involved in glycan recognition and interaction [8]. Several of the 69 classified CBM families have been found associated with PLs but no CBM family is associated exclusively with PLs. CBMs perform diverse functions related to substrate recognition [9,10], yet the role of CBMs in PL function is poorly understood. The prediction of the CBMs specificity is crucial for determining the role of the CBM and function of its associated enzyme [11]. Structures of several PLs with additional domains have been determined but none with CBMs. Poly-functional PLs, are also known, where the lyase domain is associated with another enzymatic

domain, often with complementary activities. For example, in Saccharophagus degradans 2–40, three putative alginate lyase domains (PL6_3, PL7_5 and PL6_3) are associated together within one polypeptide chain (GenBank accession number ABD82130.1). Another level of organization was found in Bacteroidetes where genes required for full degradation of specific substrates form clusters named Polysaccharides Utilization Loci (PULs) (Figure 2) [12]. Approximately 90 PULs were identified in Bacteroides thetaiotaomicron [13] and 118 Bacteroides cellulosilyticus WH2 [14]. Gene clustering of sugar-cleaving enzymes have also been observed in Firmicutes [15]. Although some PULs contain CAZymes with known activities, others contain unknown CAZymes. Participation of an unknown CAZyme in a specific PUL might help to identify its substrate.

Galacturonan lyases Pectin forms a complex network made of galacturonan and other sugars, which together with cellulose and hemi-cellulose are the main components of plant cell walls [16]. Six PL families are involved in the degradation of the poly-galacturonan regions of pectin and are classified as pectate lyases (PL1, PL2, PL3, PL9, PL10) or oligo-galacturonan lyases (PL22). The other families

Figure 2

BT4652 PL15_2

BT4657 UNK

UNK

UNK

UNK

PL12_2

BT4662

BT4658 GH88

SusD

SusC

UNK

PL12_2

BT4664 HTCS

PL13

Current Opinion in Structural Biology

An example of a PUL. Heparin PUL from Bacteroides thetaiotaomicron. UNK are hypothetical proteins or non-CAZymes. Arrows indicate gene orientation. HTCS is Hybrid Two-Component System regulator. BTNNNN are locus tag identification. Current Opinion in Structural Biology 2014, 28:87–95

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Uronic polysaccharide degrading enzymes Garron and Cygler 89

(PL4 and PL11) are rhamnogalacturonan lyases cleaving the a-1,4 bond between rhamnose and galacturonic acid (Table S1).

PL11 (rhamnogalacturonan lyases) have a b-propeller fold, albeit with eight blades, suggesting different ancestors for these two families. PL22 utilizes Mn2+ in a metalassisted mechanism. Based on the utilization of Mn2+ and active site topology Boraston and coworkers suggested that PL22 is mechanistically similar to PL2 enzymes (Figure 4 in [19]). Convergent evolution has previously been suggested for the PL1 and PL10 families [20].

Metal-assisted catalytic mechanism

All galacturonan lyases, with the exception of PL4 rhamnogalacturonan lyases, use metal-assisted mechanism to break the glycosidic bond in pectin by syn elimination (Figure 1b, Table S2). In this mechanism, a metal ion neutralizes the carboxylic group in the +1 position. The interaction between the carboxylic group and the metal ion enhances the reactivity of the C5 proton. A basic amino acid abstracts this more labile proton. The proton for the newly formed reducing end is likely provided by a water molecule [6]. The most frequently observed metal is Ca2+, likely due to its presence in cellular interspaces and its presence as counter ions in pectin. This ion is frequently found within the carbohydrate binding motifs in carbohydrate binding proteins [10,17].

Catalytic mechanism of PL4 rhamnogalacturonan lyases

PL4 is the second PL family of rhamnogalacturonan lyases and the only galacturonan lyase family that does not utilize the metal assisted-mechanism. This family displays a unique fold of three b-sandwich domains not found in other PL or GH families [21]. The structure of the rhamnogalacturonan lyase from Aspergillus aculeatus complexed with rhamnogalacturonan allowed identification of the active site and the location of 3 to +3 subsites (PDB code 3NJV, [22]). The substrate-binding site extends over two of the three domains of this enzyme. The ‘’ subsites are formed by domain III and the ‘+’ subsites are formed by domain I (Figure 3). The substrate forms hydrogen bonds with four arginines (Arg111, Arg107, Arg451 and Arg455), which are in a well-conserved segment of PL4 enzymes. Two amino acids (Lys150 and His210) are in positions consistent with the b-elimination mechanism and their importance was confirmed by mutagenesis [22]. Lys150 is located in the

PL2 and PL22 evolved to use Mn2+ instead of Ca2+. Calcium levels within the cell are highly controlled, likely diverting the intracellular lyases toward the use of Mn2+ [18]. The PL22 family contains predominantly bacterial oligo-galacturonan lyases involved in the degradation of saturated or unsaturated galacturonate disaccharides [19]. PL22 enzymes have a b-propeller fold with seven blades. This fold is rare among PLs families; only Figure 3

(a)

(b) Arg111

Arg111

Arg107

Arg107 +2

+2

+1

+1

Lys150 –1

–1 Arg451

Arg451 Arg455

Arg455 His210

Lys150

His210

Current Opinion in Structural Biology

The PL4 rhamnogalacturonan lyase from Aspergillus aculeatus K150A complexed with rhamnogalacturonan (PDB code 3NJV). (a) Cartoon representation of the enzyme. The N-terminal domain is grey, middle domain is cyan and C-terminal domain is magenta. The hexasaccharide substrate is shown in stick representation. (b) The close-up of the substrate binding site showing the key residues making contacts with the substrate. This and other figures were prepared with PyMOL (http://www.pymol.org/). www.sciencedirect.com

Current Opinion in Structural Biology 2014, 28:87–95

90 Carbohydrate–protein interactions and glycosylation

vicinity of the C5 carbon of GalA in the +1 subsite and probably plays the role of a Brønsted base, while His210 is proximal to the glycosidic oxygen and is well suited for the role of a Brønsted acid. Asp139 and two water molecules form hydrogen bonds with the carboxylic group of +1 GalA neutralizing the charge. The composition of this active site indicates a His/Tyr mechanism rather than the metal-assisted one, prevalent among oligogalacturonan lyases.

Alginate and GAG lyases GAGs are linear polysaccharides composed of disaccharide repeat units containing a hexosamine 1–4 linked to an uronic acid and are produced mainly by eukaryotes [1]. GAGs have some similarities to alginates, linear polysaccharides containing variable ratios of b-1,4-D-polymannuronate (M) and a-1,4-L-polyguluronate (G) [23]. For example, several heparin-binding proteins also bind sulfated alginate [24]. Therefore, it is not surprising to find similarities between enzymes degrading these substrates. All alginate, glycosaminoglycan or glucuronic lyases (with the exception of the PL6 family) use the same histidine/ tyrosine mechanism. Two main folds occur in both groups: an (a/a)6 toroid + b-sandwich and the b-jellyroll (Table S2). The histidine/tyrosine mechanism is well adapted to syn as well as to anti elimination. Consequently, the polysaccharide lyases utilizing this mechanism can cleave the bond next to an uronic acid and/or its C5-epimer. Despite different folds, the enzymes using this mechanism share a strikingly similar arrangement of their catalytic residues. The neutralizer — usually Glx/ Asx — is located near the carboxylic group of the uronate at the +1 subsite. Several other charged amino acids (His, Arg or Glu) assist the neutralizer through a complex network of hydrogen bonds between the enzyme and the substrate. A tyrosine, strictly conserved among all these PLs, is most likely the Brønsted acid in anti elimination while the Brønsted base during anti elimination is a histidine. In syn elimination the tyrosine plays a dual role as an acid and a base (Figure 1b, Table S2). There are other relatively well-conserved amino acids near the catalytic site, indicating their participation in the substrate degradation but their exact roles have not been unequivocally established. PL12, PL17 and PL23 adopt (a/a)6 toroid + b-sandwich fold

PL12s are heparin-sulfate lyases believed to be specific for breaking the linkage next to GlcA. Family PL17 contains only bacterial alginate lyases. Characterization of the enzyme from Sphingomonas sp. showed that this enzyme is active against polyM, polyG and polyMG regions, with preference for polyM [25]. The recently identified PL23 contains only viral enzymes, which are chondroitin lyases with preference for poorly sulfated Current Opinion in Structural Biology 2014, 28:87–95

substrates [26]. Unsulfated chondroitin is one of the main compounds found in peritrophic membranes of the insect midgut, which is the target for the virus encoding PL23 chondroitinase. The recently determined structures of enzymes from PL12, PL17 and PL23 families have the (a/a)6 toroid + b-sandwich fold. Their catalytic residues and global architecture of their active sites are similar, suggesting similarity of their catalytic mechanisms (Figure 4, Table S2). The catalytic tyrosine is strictly conserved, Tyr314 in PL12 (PDB code 4FNV [27]), Tyr258 in PL17 (PDB code 4OJZ [25]) and Tyr299 in PL23 (PDB code 3VSM [28]) (Figure 5b,c). Histidine, which plays the role of a Brønsted base during anti elimination, is present in some PL12 enzymes (e.g. His241 in Pedobacter heparinus heparinase III (code PDB 4MMH [29])), but not in others (Ile261 in Bacteroides thetaiotaomicron heparinase III (code PDB 4FNV)); it is present in PL17 enzymes (His202 in Saccharophagus degradans 2–40 alginate lyase (PDB code 4OJZ [25])) but not in PL23 enzymes. This correlates well with the stereospecificity of activity: PL23 and PL12 perform syn elimination whereas PL17 can perform both. The presence of a histidine in some bacterial strains in the PL12 family suggests a possibility for anti elimination as well. In addition to the catalytic amino acids, there is substantial overall similarity between these families. For example, the PL17 enzyme Alg17c (PDB code 4OJZ) contains a structural Zn2+ ion that stabilizes long loops from the b-sandwich domain, which form part of the substrate binding site. A Zn2+ ion is found in a similar position in heparinase II (family PL21, PDB code 2FUT [30]) with the same coordination, Heparinase C from P. heparinus (family PL12, PDB code 4MMH) has a Ca2+ ion in the same position, also with similar coordination. This site is also conserved in the structure of heparinase III from Bacteroides thetaiotaomicron (PDB code 4FNV), although it is unoccupied. Chondroitinase AC from Arthrobacter aurescens belonging to PL8 (PDB code 1RW9 [31]) has a Na+ ion close to the active site but not in the same position as in PL12, PL17 and PL21. The recent structures of PL12 enzymes confirm the plasticity of the (a/a)6 toroid and b-sandwich domains previously observed in PL8 [32]. Heparinase III from P. heparinus (PDB code 4MMH) has an open conformation with the neutralizer Asn240 positioned 10 A˚ from the catalytic Tyr294, an arrangement clearly incompatible with catalysis [29]. B. thetaiotaomicron heparinase III (PDB code 4FNV) has a more compact conformation [27]. The first PL23 structure also displays an open conformation where the neutralizer Asn236 is apparently too far from the catalytic residues [28] (Figure 5c). This observed flexibility might be necessary for accommodating the substrates in the enzyme’s binding site. www.sciencedirect.com

Uronic polysaccharide degrading enzymes Garron and Cygler 91

Figure 4

PL12, 4FNV

PL17, 4NEI

PL23, 3VSM Current Opinion in Structural Biology

Cartoon representation of enzymes from families PL12, PL17 and PL23 sharing the (a/a)6 toroid + b-sandwich fold. The proteins are rainbow colored, from blue at the N-terminus to red at the C-terminus.

Common evolutionary origins of alginate and GAG lyases

The evolutionary tree based on structural comparison of the six families having the (a/a)6 toroid plus b-sandwich fold shows two distinct evolutionary branches (Figure 5a). Based on substrate specificity, it was expected that GAG lyases (PL8, PL12, PL21 and PL23) would compose one branch of the tree while alginate lyases (PL15 and PL17) the second branch. However, the tree shows that GAG lyases PL12 and PL21 are closer to alginate lyase PL15 and PL17 than to PL8 and PL23. The close distance between viral PL23 and bacterial PL8 suggests a horizontal gene transfer of chondroitin lyases between bacteria and viruses. The only other viral GAGs lyases belong to PL16 and have a triple stranded b-helix fold [33]. This particular organization is also found for the viral K5 lyase (non classified lyase), which cleaves capsular polysaccharide containing GlcA [34]. This fold is common to several viral tail fibre proteins and suggests the possibility of a common evolutionary origin for these two functions. The evolutionary link between alginate and GAG lyases extends to others folds. Heparinase I from PL13 with a bjellyroll fold is structurally similar to PL7 and PL18 alginate lyases. Recently, hyaluronan lyase activity has been found in a PL5 alginate lyase with the (a/a)6 toroid fold [35]. Moreover, b-helix PL6 lyases utilizing the www.sciencedirect.com

metal-assisted mechanism contain both alginate lyases and chondroitinase B. Since alginate preceded GAGs during evolution, the presence of the same folds in GAG and alginate lyases suggest that GAG lyases arrived by a divergent evolution from alginate lyases.

Polysaccharide lyases versus polyuronic glycoside hydrolases Hydrolysis is used more frequently than b-elimination for polysaccharide degradation with GHs comprising 57% of the CAZymes ‘mini-microbiome’ while PLs represent only 2% [15]. However, out of 133 GH families only four GH families contain GAG-degrading enzymes and some members of the GH28 family hydrolyze pectin. No hydrolases are known to degrade alginate. It is noteworthy that the majority of GAG hydrolases are from eukaryotes while prokaryotes utilize principally the lytic mechanism. b-Helix fold in pectin-degrading PL families and GH28

A right-handed b-helix fold is common to GH and PL families and it was suggested that they evolved from a common ancestor [36]. The b-helix is found in four PL families and in seven GH families. The most interesting is comparison of pectin-degrading PLs and the GH28 family. GH28 are inverting enzymes, using a classical single displacement mechanism involving three acidic Current Opinion in Structural Biology 2014, 28:87–95

92 Carbohydrate–protein interactions and glycosylation

Figure 5

(a)

PL12 4MMH PL17 4NEI PL15 3A0O PL21 2FUQ PL8ABC 2Q1F PL23 3VSM PL8HL 1F1S PL8XL 1J0M PL8AC 1RW9 PL8ACped 1HM2

(b)

(c)

Tyr341/258

Tyr299/242 Glu309/254 Glu395/407

Tyr490/450

His291/233

Asn260/201 His464/413

lle261

Asn183 Arg345/296

Asn236

+1

+2 His202

Current Opinion in Structural Biology

(a) Evolutionary tree of alginate and GAGs lyases with the (a/a)6 toroid + b-sandwich fold. A representative structure for each PL family was selected. Matrix distance, using Kendall’s correlation [49], was calculated based on PDBeFOLD Q-score (http://www.ebi.ac.uk/msd-srv/ssm/), which compares folds and sequence similarities. The FastMe algorithm was used to build the tree [50]. Each PL family is followed by the PDB code used to create this matrix. Several PL8 enzymes with different functions were selected: ABC is for chondroitinase ABC, HL for hyaluronan lyase, XL for xanthan lyase, AC for chondroitinase AC, ACped for chondroitinase AC belonging to Pedobacter heparinus. Statistic matrix is on supplementary data. (b) Superposition of PL12 in grey (4FNV) with PL17 in yellow (4OJZ). Conserved amino acids of the active sites are represented in sticks; catalytic tyrosine is indicated in red, the numbering of amino acids is respectively PL12/PL17. (c) Superposition of PL23 in blue (3VSM) with PL8 in green (1RWH). Conserved amino acids of the active sites are represented in sticks; catalytic tyrosine is indicated in red, the numbering of amino acids is respectively PL23/PL8. PL23 is in open conformation.

amino acids; one of them plays the role of a general acid whereas the other two act as general bases [37]. The topological assignment of b-strands differs slightly but the structures superimpose very well and the typical structural features of the b-helix are conserved (Figure 6a). Moreover, the substrate subsites are in similar positions in both enzymes although the substrates bind in opposite directions (Figure 6b). This fold is also found in pectin methylesterases belonging to family 8 of carbohydrate esterases (CE8). Such close fold similarity and similar location of the substrate suggest indeed that the b-helix fold is well suited to pectin binding and was able to acquire different arrangements of catalytic residues to degrade pectin through different mechanisms. Current Opinion in Structural Biology 2014, 28:87–95

TIM barrel fold in GAG hydrolases

The GAG hydrolases have the TIM barrel fold. This fold has not been observed among PLs. GH2, GH39 and GH79 have the (b/a)8 TIM barrel fold (clan GH-A) and are retaining enzymes degrading b-glycosidic bond and contain ‘uronic’ hydrolases, which bind uronic acids in the 1 subsite. Enzymes with b-glucuronidase activity (EC 3.2.1.31) are also found in other GH families, for example, GH1 and GH30, but no activity toward GAGs has yet been demonstrated. The GH56 enzymes have the (b/a)7 TIM barrel fold and are hexosaminidases. Like PLs, they accommodate uronic acid in +1 subsite and cleave the N-acetyl-hexosaminidase-b-1,4-uronic acid bond, with specificity for chondroitin sulfate and hyaluronan. www.sciencedirect.com

Uronic polysaccharide degrading enzymes Garron and Cygler 93

Figure 6

(a)

(b)

–1

Asp381

+1

–2

–1

+2

+3

Arg218

Asp402

Asp403

Current Opinion in Structural Biology

Structural similarity of pectin acting polysaccharide lyases and glycoside hydrolases with the b-helix fold PL1 enzyme (PDB code 1AIR) is colored green and GH28 (2UVF) in pink. (a) Cartoon representation of these two enzymes; (b) superposition of their active sites with bound substrates. The PL1 tetrasaccharide from the R218L mutant (2EWE) was docked on WT (1AIR). The tetrasaccharide occupies 1 to +3 subsites. The disaccharide product in GH28 occupies 1 and 2 subsites. Catalytic residues are shown in stick representation.

Eukaryotic GAGs hydrolases are involved in numerous pathologies (e.g. lysosomal storage diseases) and are widely studied [38]. Structures of representative human enzymes are available for GH2 [39], GH39 [40,41] and GH56 [42]. GH79 contains examples of human heparanases and hyaluronoglucuronidases. Heparanases are involved in cancer metastasis and cellular processes such as inflammation and the blood coagulation cascade [43]. The only known structure in this family is of a plant bglucuronidase from Acidobacterium capsulatum [44]. Unfortunately, this structure does not clarify the details of GAG hydrolysis by eukaryotic GH79 enzymes. The structure of heparanase, which is secreted as pre-pro-heparanase and is cleaved into two parts (8 kDa + 50 kDa) to form an active enzyme [45], remains a challenge.

Summary and future prospects of PLs Whatever the origin or the fold of the lyases, two main enzymatic mechanisms have emerged: the metal-assisted mechanism, specific to pectin substrate cleavage, and the His/Tyr for syn and anti b-elimination of various other substrates. The majority of the 23 PL families are dedicated to degradation of three main polysaccharides containing uronic acid, namely pectin, alginate and GAGs.

was also detected in Bacillus sp. GL1 [48]. The structures of ulvan or gellan lyases are still unknown and it will be interesting to see if these two activities fall into one of the two mechanisms common so far to all PLs. Uronic acids are frequently found in the polysaccharides of bacterial capsules. The capsule is essential for protecting bacteria but also aids in virulence. The capsular polysaccharide is also essential for biofilm formation, helping bacteria to survive in hostile environments. Many bacteria synthesize unique uronic polysaccharides and specialized enzymes that are required for their degradation must exist and have to be identified. The many ongoing genomic and metagenomic projects will quickly increase the number of known sequences identifiable as CAZymes and should help the discovery of new PL families in the future. Many PL subfamilies have no characterized enzymes and much work will be needed to fill the gaps. Another large area of future research will be to define the activities and structures of the ‘non classified’ enzymes and should become one of the most interesting subjects of PL research. Finally, the new PULs will help in understanding their roles and their function in the life cycle of the organisms that contain them within their genomes.

Acknowledgements Over 250 enzyme sequences within CAZy are ‘non classified’ polysaccharide lyases, undetectably or very distantly related to the classified PL families. Only three of them have been enzymatically characterized and shown to degrade ulvan [46], alginate [47] and K5 glycosaminoglycan polysaccharide [34]. In addition, gellan lyase activity www.sciencedirect.com

We would like to thank all the CAZy team and especially N. Lenfant for the evolution tree, N. Terrapond for the PULs studies, P. Coutinho, B. Henrissat and J. Leefor their comments and advice and D. Palmer for help with chemical diagrams. This work supported by National Science and Engineering Research Council (NSERC) to MC and by Aix-Marseille Universite´. Current Opinion in Structural Biology 2014, 28:87–95

94 Carbohydrate–protein interactions and glycosylation

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.sbi. 2014.07.012.

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Mikami T, Kitagawa H: Biosynthesis and function of chondroitin sulfate. Biochim Biophys Acta 2013, 1830:4719-4733.

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Henrissat B: A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1991, 280(Pt 2):309-316.

4.

Campbell JA, Davies GJ, Bulone V, Henrissat B: A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem J 1997, 326(Pt 3):929-939.

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Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B: The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 2014, 42:D490-D495. This paper describes the recent advances in the CAZy database.

6. 

Garron ML, Cygler M: Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology 2010, 20:1547-1573. This review summarized structural knowledge of the PL families, identified the number of different PL folds, classified catalytic mechanisms into two categories indicating convergent evolution of PLs.

7. 

Lombard V, Bernard T, Rancurel C, Brumer H, Coutinho PM, Henrissat B: A hierarchical classification of polysaccharide lyases for glycogenomics. Biochem J 2010, 432:437-444. This paper analyzed CAZy families and subdivided them based on phylogenetic analysis into subfamilies that correlate well with substrate specificity. 8.

9.

Boraston AB, Bolam DN, Gilbert HJ, Davies GJ: Carbohydratebinding modules: fine-tuning polysaccharide recognition. Biochem J 2004, 382:769-781. Montanier C, van Bueren AL, Dumon C, Flint JE, Correia MA, Prates JA, Firbank SJ, Lewis RJ, Grondin GG, Ghinet MG et al.: Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function. Proc Natl Acad Sci U S A 2009, 106:3065-3070.

10. Gilbert HJ, Knox JP, Boraston AB: Advances in understanding  the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Curr Opin Struct Biol 2013, 23:669-677. This review presents functional classification of CBMs and proposes a novel classification of cell walls CBMs based on their interactions with cellulose polysaccharides.

diet on resource utilization by a model human gut microbiota containing Bacteroides cellulosilyticus WH2, a symbiont with an extensive glycobiome. PLoS Biol 2013, 11:e1001637. 15. El Kaoutari A, Armougom F, Gordon JI, Raoult D, Henrissat B: The  abundance and variety of carbohydrate-active enzymes in the human gut microbiota. Nat Rev Microbiol 2013, 11:497-504. This paper presents a thorough comparative analysis of CAZymes in various phyla present in human gut based on the large number of sequenced genomes. 16. Palin R, Geitmann A: The role of pectin in plant morphogenesis. Biosystems 2012, 109:397-402. 17. Taylor ME, Drickamer K: Convergent and divergent mechanisms of sugar recognition across kingdoms. Curr Opinion in Struct Biol 2014, 28:14-22 http://dx.doi.org/10.1016/ j.sbi.2014.07.003. 18. Abbott DW, Boraston AB: Structural biology of pectin degradation by Enterobacteriaceae. Microbiol Mol Biol Rev  2008, 72:301-316. This paper presents a comparison between the enzymes using Mn2+ assisted mechanism in PL22 and PL2 and hypothesizes on the relationship between cellular distribution of enzymes and metal used for their catalytic mechanism. 19. Abbott DW, Gilbert HJ, Boraston AB: The active site of oligogalacturonate lyase provides unique insights into cytoplasmic oligogalacturonate b-elimination. J Biol Chem 2010, 285:39029-39038. 20. Charnock SJ, Brown IE, Turkenburg JP, Black GW, Davies GJ:  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 2002, 99:12067-12072. This paper describes the first example of convergent evolution among the PL enzyme families. 21. McDonough MA, Kadirvelraj R, Harris P, Poulsen JC, Larsen S: Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4. FEBS Lett 2004, 565:188-194. 22. Jensen MH, Otten H, Christensen U, Borchert TV, Christensen LL, Larsen S, Leggio LL: Structural and biochemical studies elucidate the mechanism of rhamnogalacturonan lyase from Aspergillus aculeatus. J Mol Biol 2010, 404:100-111. 23. Hay ID, Ur Rehman Z, Moradali MF, Wang Y, Rehm BH: Microbial alginate production, modification and its applications. Microb Biotechnol 2013, 6:637-650. 24. Freeman I, Kedem A, Cohen S: The effect of sulfation of alginate hydrogels on the specific binding and controlled release of heparin-binding proteins. Biomaterials 2008, 29:3260-3268. 25. Park D, Jagtap S, Nair SK: Structure of a PL17 family alginate  lyase demonstrates functional similarities among exotype depolymerases. J Biol Chem 2014, 289:8645-8655. This paper presents the first structural and biochemical characterization of an enzyme from the PL17 family. Structure of the enzyme–substrate complex and mutagenesis identified the catalytic machinery. 26. Sugiura N, Ikeda M, Shioiri T, Yoshimura M, Kobayashi M, Watanabe H: Chondroitinase from baculovirus Bombyx mori nucleopolyhedrovirus and chondroitin sulfate from silkworm Bombyx mori. Glycobiology 2013, 23:1520-1530.

11. Abbott DW, Lammerts van Bueren A: Using structure to inform  carbohydrate binding module function. Curr Opinion Struct Biol 2014, 28:32-40 http://dx.doi.org/10.1016/j.sbi.2014.07.004. This review summarizes Carbohydrate Binding Domains functions and potential for prediction their function from structure.

27. Dong W, Lu W, McKeehan W, Luo Y, Ye S: Structural basis of  heparan sulfate-specific degradation by heparinase III. Protein Cell 2012, 3:950-961. This paper reported the first structure of heparan sulfate lyase from the PL12 family.

12. Bjursell MK, Martens EC, Gordon JI: Functional genomic and metabolic studies of the adaptations of a prominent adult human gut symbiont, Bacteroides thetaiotaomicron, to the suckling period. J Biol Chem 2006, 281:36269-36279.

28. Kawaguchi Y, Sugiura N, Kimata K, Kimura M, Kakuta Y: The  crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein. FEBS Lett 2013, 587:3943-3948. This paper described the first structure of an enzyme from the PL23 family. Catalytic amino acids were idenitfied by comparison with PL8 enzymes.

13. Martens EC, Chiang HC, Gordon JI: Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell Host Microbe 2008, 4:447-457. 14. McNulty NP, Wu M, Erickson AR, Pan C, Erickson BK, Martens EC, Pudlo NA, Muegge BD, Henrissat B, Hettich RL et al.: Effects of Current Opinion in Structural Biology 2014, 28:87–95

29. Hashimoto W, Maruyama Y, Nakamichi Y, Mikami B, Murata K: Crystal structure of pedobacter heparinus heparin lyase Hep III with the active site in a deep cleft. Biochemistry 2014, 53:777-786. www.sciencedirect.com

Uronic polysaccharide degrading enzymes Garron and Cygler 95

30. Shaya D, Tocilj A, Li Y, Myette J, Venkataraman G, Sasisekharan R, Cygler M: Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product. J Biol Chem 2006, 281:15525-15535.

40. Maita N, Tsukimura T, Taniguchi T, Saito S, Ohno K, Taniguchi H, Sakuraba H: Human alpha-L-iduronidase uses its own Nglycan as a substrate-binding and catalytic module. Proc Natl Acad Sci U S A 2013, 110:14628-14633.

31. Lunin VV, Li Y, Linhardt RJ, Miyazono H, Kyogashima M, Kaneko T, Bell AW, Cygler M: High resolution crystal structure of arthrobacter aurescens chondroitin ac lyase: enzyme– substrate complex defines the catalytic mechanism. J Mol Biol 2004, 337:367-386.

41. Bie H, Yin J, He X, Kermode AR, Goddard-Borger ED, Withers SG, James MNG: Insights into mucopolysaccharidosis I from the structure and action of a-L-iduronidase. Nat Chem Biol 2013, 9:739-745.

32. Elmabrouk ZH, Vincent F, Zhang M, Smith NL, Turkenburg JP, Charnock SJ, Black GW, Taylor EJ: Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations. Proteins: Struct Funct Bioinform 2011, 79:965-974. 33. Smith NL, Taylor EJ, Lindsay AM, Charnock SJ, Turkenburg JP, Dodson EJ, Davies GJ, Black GW: Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix. Proc Natl Acad Sci U S A 2005, 102:17652-17657. 34. Thompson JE, Pourhossein M, Waterhouse A, Hudson T, Goldrick M, Derrick JP, Roberts IS: The K5 lyase KflA combines a viral tail spike structure with a bacterial polysaccharide lyase mechanism. J Biol Chem 2010, 285:23963-23969. 35. MacDonald LC, Berger BW: A polysaccharide lyase from Stenotrophomonas maltophilia with a unique, pH-regulated substrate specificity. J Biol Chem 2014, 289:312-325. 36. Jenkins J, Pickersgill R: The architecture of parallel beta-helices and related folds. Prog Biophys Mol Biol 2001, 77:111-175. 37. van Santen Y, Benen JA, Schroter KH, Kalk KH, Armand S, Visser J, Dijkstra BW: 1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. J Biol Chem 1999, 274:30474-30480. 38. Matte U, Yogalingam G, Brooks D, Leistner S, Schwartz I, Lima L, Norato DY, Brum JM, Beesley C, Winchester B et al.: Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients. Mol Genet Metab 2003, 78:37-43. 39. Jain S, Drendel WB, Chen ZW, Mathews FS, Sly WS, Grubb JH: Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs. Nat Struct Biol 1996, 3:375-381.

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42. Chao KL, Muthukumar L, Herzberg O: Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 2007, 46:69116920. 43. Vlodavsky I, Beckhove P, Lerner I, Pisano C, Meirovitz A, Ilan N, Elkin M: Significance of heparanase in cancer and inflammation. Cancer Microenviron 2012, 5:115-132. 44. Michikawa M, Ichinose H, Momma M, Biely P, Jongkees S, Yoshida M, Kotake T, Tsumuraya Y, Withers SG, Fujimoto Z et al.: Structural and biochemical characterization of glycoside hydrolase family 79 beta-glucuronidase from Acidobacterium capsulatum. J Biol Chem 2012, 287:14069-14077. 45. Levy-Adam F, Miao HQ, Heinrikson RL, Vlodavsky I, Ilan N: Heterodimer formation is essential for heparanase enzymatic activity. Biochem Biophys Res Commun 2003, 308:885-891. 46. Nyvall Collen P, Sassi JF, Rogniaux H, Marfaing H, Helbert W: Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family. J Biol Chem 2011, 286:42063-42071. 47. Lundqvist LCE, Jam M, Barbeyron T, Czjzek M, Sandstro¨m C: Substrate specificity of the recombinant alginate lyase from the marine bacteria Pseudomonas alginovora. Carbohydr Res 2012, 352:44-50. 48. Miyake O, Kobayashi E, Nankai H, Hashimoto W, Mikami B, Murata K: Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1. Arch Biochem Biophys 2004, 422:211-220. 49. de Hoon MJ, Imoto S, Nolan J, Miyano S: Open source clustering software. Bioinformatics 2004, 20:1453-1454. 50. Desper R, Gascuel O: Fast and accurate phylogeny reconstruction algorithms based on the minimum-evolution principle. J Comput Biol 2002, 9:687-705.

Current Opinion in Structural Biology 2014, 28:87–95

Uronic polysaccharide degrading enzymes.

In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polys...
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