Polysaccharide Lyase Family 7

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• Authors: ^^^Nadine Gerlach^^^ and ^^^Jan-Hendrik Hehemann^^^
• Responsible Curator: ^^^Jan-Hendrik Hehemann^^^

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Polysaccharide Lyase Family PL7
3D Structure	β jelly roll
Mechanism	β-elimination
Active site residues	known
CAZy DB link
https://www.cazy.org/PL7.html

Mechanism

Figure 1. Multiple protein alignment of Aly PL7 as well as a secondary structure prediction of the crystallized PL7 from Klabsiella pneumoniae (PDB code: 4OXZ, [1]). Conserved residues in the homologues are colored in red and (putative) catalytic residues are indicated by a star. MSA was done using Espript3.0 [2].

Alginate lyases (Alys) of all families, PL5-7, PL14-15, PL17-18, catalyse degradation of the anionic alginate in three steps: (I) removal of the negative charge on the carboxylate anion, (II) general base-catalysed abstraction of the proton on the C5 and (III) β-elimination of the 4-O-glycosidic bond [3]. Most PL7s are endo-active, i.e. acting within a poly- or oligosaccharide and releasing smaller alginate fragments, while exo-acting PL7s are cleaving a monosaccharide from the polymer termini [4]. In both modes of action, a new non-reducing end with a 4-deoxy-L-erythro-hex-4 en pyranosyl uronate residue (Δ) is formed. In contrast to terrestrial PL7, marine PLs need the divalent cation calcium for substrate recognition and binding [4]. Ca²⁺ is weakening the ionic interactions between substrate (polyanion) and PL7 (polycation) by reducing the surface density of the alginate charge and therefore increasing the enzyme activity [5].

Kinetics and catalytic residues

Several structural and biochemical analyses of wild type and mutated PL7s revealed five residues forming the active site: arginine (R), glutamine (Q), histidine (H), tyrosine (Y) [6][7][8], which are assembled in three highly conserved regions: R*ELR*ML, VIIGQ(I/V)H, YFKAG*Y*Q (Figure 1) [9]. Osawa and colleagues assumed that in PL7 ALY-1 from Corynebacterium sp. Q117+Y195 interact near the reaction site of alginate to maintain proper orientation of the substrate, R72 interacts with alginate due to the formation of salt bridges with the carboxyl groups at the C5 and H119 acts as a base to deprotonate [10]. However, there can also be additional charged residues at the active site, which promote substrate recognition and binding [4]. Such residues can be found in the N-terminal R*ELREML and VIIGQIH regions. Both highly conserved regions are mainly characterized by hydrophobic amino acids (especially aromatic amino acids) such as leucine, tryptophan and methionine as well as residues with planar polar side chains (especially amino acids with charged side chains) such as arginine, glutamic acid, glutamine (Figure 2). These residues have been suggested to be substrate-binding molecules [9].

Substrate specificities

Figure 2. Subfamilies of PL7s [4].

Polysaccharide lyase family 7 (PL7) contains five subfamilies (SF) based on their sequence similarities [11], plus a so far uncharacterized sixth subfamily, which consist only of marine representatives of the Flavobacteriaceae (Figure 2) [4]. All characterized PL7 enzymes are alginate lyases specific for the anionic, gel forming polysaccharide alginate. The substrate specificity depends on the source of alginate, i.e. derived from brown seaweed or mucoid bacteria Pseudomonas spp. and Azotobacter vinelandii, as well as geographical and saisonal parameters. Alginate is an heteropolysaccharide, consisting of β-D-mannuronate (M) and α-L-guluronate (G). These monosaccharides can occur in homogenous and heterogenous blocks. Hence, PL7 lyases can be mannuronate (EC 4.2.2.3), guluronate (EC 4.2.2.11) or mixed link (EC 4.2.2.-) specific. Despite the preference for M- or G-enriched blocks, most PL7s also have a moderate to low processivity for the other building block [4][12][13]. PolyG specific PL7s have been found in the SF3 and SF5 [4] with QIH in the second highly conserved region, while polyM specific PL7s are characterised by QVH [14][15].

Three-dimensional structures

Figure 3. 3D Structure of endo- and exo-active PL7s [4]. (A,B) endo AlyA1 and (C, D) exo AlyA5 from Zobellia galaactinovorans DsijT shown as cartoon (A,C) and surface structure (B,D) with superimposed tetrasaccharide from PDB:2ZAA [16]. The image was conducted in PyMOL [17].

PL 7 is a well biochemical characterized family with almost 40 entries in the CAZy data base [18]. Structural insights on the other hand are still limited to nine 3D structures from only eight bacterial strains (status at CAZy in August 2019). The first structure of a PL7 was determined from Pseudomonas aeruginosa by multiple isomorphous replacement (MIR) at 2.0 Å resolution [7]. Like PL14, PL7 belongs to the jelly roll family with a wide open cleft harboring the active site (Figure 3A, B). Til date, there is only one known exoactive PL7 structure [4]. Zobellia galaactinovorans DsijT is harboring, among others, two PL7 with two completely different activity modifs. AlyA1 belongs to SF3 and is an endo-active PL7. AlyA5 on the other hand belongs to SF5 and is exo-active, which active site is close by three additional loops forming a small pocket (Figure 3C, D). The highly conserved 9-amino-acid-block YFKAGVY*Q (where * is a variable residue) at the C-terminus of PL7s (Figure 2) has also been found for an extracellular pectate lyase in E. chrysanthemi (Keen & Tamaki, 1986) . Alginate and pectate / pectin lyases share several features such as β-elimination, the recognition of substrates of a similar structure and primary sequence similarity, indicating that they probably share a similar core structural fold. Since alginate lyases and pectinases differ in substrate specificity, it is likely not related to substrate recognition, but rather to maintaining a stable 3D-conformation [9]. Several alginate lyases have been reported to be multimodular domain enzymes with putative non-catalytic, carbohydrate-binding modules (CBMs). However, their exact role has been barely investigated yet. The first biochemically characterized CBM of an endo PL7 was a N-terminal CBM13 from Agarivorans sp. L11, which not only increased its substrate binding ability and therefore catalytic efficiency, but also substrate preference and product profiling as well as thermostability [19]. Contradictory observations regarding catalytic effiency and substrate specificity were made for an endo PL7 from Vibrio splendidus OU02 DNA with an N-terminal CBM32 linked by a unique alpha helix linker [20]. Nevertheless, the CBM and und linker were supposed to serve as "pivont point" affecting the product distribution towards trisaccharides. The PL7 from Persicobacter sp. CCB-QB2 is even consisting of three domains - a N-terminal CBM16 with still unclear function, C-terminal catalytic domain and a CBM32, which is located between both domains [13]. This CBM32 is also not enhancing the catalytic activity and is not binding alginate, but the cleaved termini during catalysis. The crystal structures revealed an arginine residue which is possibly binding to the carboxylic group and a conserved Ca²⁺ binding site being most likely essential for the maintenance of the overall fold.

Gene transfer of Alys among different habitats

Alginate degrading organisms often posses specified gene clusters for glycan utilization which contain, among other proteins such as transporters, several endo- and exo-actining Alys of different families. These gene clusters are called polysaccharide utilization loci (PULs) for Bacteriodetes [21] or alginolytic operons in case a SusCD pair transporter is missing or replaced by a different transporter system. It has been shown that these gene cluster can be transfered horizontally from one organism to another and thereby even cross different environmental habitats [22]. The first alginate utilization system (AUS) was found in Zobellia galactinovorans which contains two clusters harboring five out of seven Alys (3 PL7s). Those operons originated from an ancestral marine Flavobacterium and were independently transferred to marine Proteobacteria and Japanese gut Bacteriodetes by lateral gene transfer (LGT) [23].

Family Firsts

First catalytic endo-activity

polyM PL7 from Photobacterium ATCC 433367 [24]

polyG PL7 from Klebsiella pneumoniae subbsp. aerogenes [25]

First catalytic exo-activity

AlyA5 from Zobellia galactanivorans DsijT [4]

First 3-D apo-structure

PA1167 from Pseudomonas aeruginosa [7]

First 3-D holo-structure

A1-II from Sphingomons sp. A1 [16]

First characterised CBM

AlyL2 containing a N-terminal CBM13 from Agarivorans sp. L11 [19]

References

1. Crystal Structure of the alginate lyase from Klebsiella pneumoniae

2. Robert X and Gouet P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014;42(Web Server issue):W320-4. DOI:10.1093/nar/gku316 |

3. Alginate‐modifying enzymes: A proposed unified mechanism of action for the lyases and epimerases

4. Thomas F, Lundqvist LC, Jam M, Jeudy A, Barbeyron T, Sandström C, Michel G, and Czjzek M. (2013). Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct modes of action and exquisite adaptation to their natural substrate. J Biol Chem. 2013;288(32):23021-37. DOI:10.1074/jbc.M113.467217 |
5. Favorov VV, Vozhova EI, Denisenko VA, and Elyakova LA. (1979). A study of the reaction catalysed by alginate lyase VI from the sea mollusc, Littorina sp. Biochim Biophys Acta. 1979;569(2):259-66. DOI:10.1016/0005-2744(79)90061-5 |
6. Preston LA, Wong TY, Bender CL, and Schiller NL. (2000). Characterization of alginate lyase from Pseudomonas syringae pv. syringae. J Bacteriol. 2000;182(21):6268-71. DOI:10.1128/JB.182.21.6268-6271.2000 |
7. Yamasaki M, Moriwaki S, Miyake O, Hashimoto W, Murata K, and Mikami B. (2004). Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7: a novel alginate lyase with a beta-sandwich fold. J Biol Chem. 2004;279(30):31863-72. DOI:10.1074/jbc.M402466200 |
8. Yamasaki M, Ogura K, Hashimoto W, Mikami B, and Murata K. (2005). A structural basis for depolymerization of alginate by polysaccharide lyase family-7. J Mol Biol. 2005;352(1):11-21. DOI:10.1016/j.jmb.2005.06.075 |
9. Wong TY, Preston LA, and Schiller NL. (2000). ALGINATE LYASE: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol. 2000;54:289-340. DOI:10.1146/annurev.micro.54.1.289 |
10. Osawa T, Matsubara Y, Muramatsu T, Kimura M, and Kakuta Y. (2005). Crystal structure of the alginate (poly alpha-l-guluronate) lyase from Corynebacterium sp. at 1.2 A resolution. J Mol Biol. 2005;345(5):1111-8. DOI:10.1016/j.jmb.2004.10.081 |
11. Lombard V, Bernard T, Rancurel C, Brumer H, Coutinho PM, and Henrissat B. (2010). A hierarchical classification of polysaccharide lyases for glycogenomics. Biochem J. 2010;432(3):437-44. DOI:10.1042/BJ20101185 |
12. Badur AH, Jagtap SS, Yalamanchili G, Lee JK, Zhao H, and Rao CV. (2015). Alginate lyases from alginate-degrading Vibrio splendidus 12B01 are endolytic. Appl Environ Microbiol. 2015;81(5):1865-73. DOI:10.1128/AEM.03460-14 |
13. Sim PF, Furusawa G, and Teh AH. (2017). Functional and Structural Studies of a Multidomain Alginate Lyase from Persicobacter sp. CCB-QB2. Sci Rep. 2017;7(1):13656. DOI:10.1038/s41598-017-13288-1 |
14. Zhu B and Yin H. (2015). Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered. 2015;6(3):125-31. DOI:10.1080/21655979.2015.1030543 |
15. Deng S, Ye J, Xu Q, and Zhang H. (2014). Structural and functional studies on three alginate lyases from Vibrio alginolyticus. Protein Pept Lett. 2014;21(2):179-87. DOI:10.2174/09298665113206660094 |

16. Crystal Structure of Family 7 Alginate Lyase A1-II' H191N/Y284F in Complex with Substrate (GGMG)

17. Pymol: An open-source molecular graphics tool. CCP4 Newsletter On Protein Crystallography, 40, 82-92

18. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, and Henrissat B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014;42(Database issue):D490-5. DOI:10.1093/nar/gkt1178 |
19. Li S, Yang X, Bao M, Wu Y, Yu W, and Han F. (2015). Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution. FEMS Microbiol Lett. 2015;362(10). DOI:10.1093/femsle/fnv054 |
20. Lyu Q, Zhang K, Zhu Q, Li Z, Liu Y, Fitzek E, Yohe T, Zhao L, Li W, Liu T, Yin Y, and Liu W. (2018). Structural and biochemical characterization of a multidomain alginate lyase reveals a novel role of CBM32 in CAZymes. Biochim Biophys Acta Gen Subj. 2018;1862(9):1862-1869. DOI:10.1016/j.bbagen.2018.05.024 |
22. Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, and Michel G. (2010). Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature. 2010;464(7290):908-12. DOI:10.1038/nature08937 |
23. Thomas F, Barbeyron T, Tonon T, Génicot S, Czjzek M, and Michel G. (2012). Characterization of the first alginolytic operons in a marine bacterium: from their emergence in marine Flavobacteriia to their independent transfers to marine Proteobacteria and human gut Bacteroides. Environ Microbiol. 2012;14(9):2379-94. DOI:10.1111/j.1462-2920.2012.02751.x |
24. Malissard M, Duez C, Guinand M, Vacheron MJ, Michel G, Marty N, Joris B, Thamm I, and Ghuysen JM. (1993). Sequence of a gene encoding a (poly ManA) alginate lyase active on Pseudomonas aeruginosa alginate. FEMS Microbiol Lett. 1993;110(1):101-6. DOI:10.1111/j.1574-6968.1993.tb06302.x |
25. Baron AJ, Wong TY, Hicks SJ, Gacesa P, Willcock D, and McPherson MJ. (1994). Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes: gene cloning, sequence analysis and high-level production in Escherichia coli. Gene. 1994;143(1):61-6. DOI:10.1016/0378-1119(94)90605-x |
26. Sonnenburg ED, Zheng H, Joglekar P, Higginbottom SK, Firbank SJ, Bolam DN, and Sonnenburg JL. (2010). Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations. Cell. 2010;141(7):1241-52. DOI:10.1016/j.cell.2010.05.005 |