Polysaccharide Lyase Family 6

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• Author: Emil G.P. Stender
• Responsible Curator: ^^^Birte Svensson^^^

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Polysaccharide Lyase Family 6
3D structure	parallel β-helix
Mechanism	β-elimination
Charge neutralizer	calcium or water
Active site residues	known
CAZy DB link
https://www.cazy.org/PL6.html

Substrate specificities

PL6 contains 3 subfamilies [1] all of which contain members catalyzing the depolymerisation of alginate [2]. Alginate consisting of 1,4 linked β-D-mannuronic acid and α-L-guluronic acid arranged in poly-mannuronic acid blocks, poly-guluronic acid blocks or poly-mannuronic/guluronic acid blocks [3, 4]. Subfamily 2 and 3 have so far only shown specificity for poly-mannuronic/guluronic acid blocks [2], while subfamily 1 has been demonstrated to depolymerize poly-guluronic acid [5, 6], poly-mannuronic acid [7], poly-mannuronic/guluronic acid [2] as well as dermatan sulfate (formerly chrondroitin B) [2, 8, 9].

Kinetics and Mechanism

Figure 1. Syn – or anti – β-elimination catalyzed by PL6 enzymes acting on alginate. M represents mannuronic acid and G guluronic acid. n represents the continued sugar chain.

The β-elimination catalyzed by the PL6 enzymes results in the formation of a C4-C5 unsaturated sugar at the new non-reducing end. The first step is the neutralization of the acid group in the +1 subsite by a calcium [6, 9] or by water [5]. This lowers the pK_a value of the C5-proton allowing for abstraction by the catalytic base (Figure 1). A catalytic acid then donates a proton to the glycosidic linkage resulting in the β-elimination. This can be done in syn with the acid and base on the same side of the sugar ring in the transition state (the case for D-mannuronic acid) or anti where they are on opposite sides of the sugar ring (the case for L-guluronic acid) [10, 11].

Catalytic Residues

After charge neutralization a lysine functions as the catalytic base and an arginine the acid. They were originally identified as K253 and R273 in chondroitinase B from Pedobacter Heparinus [8]. PL6 is the only alginate lyase family to date that uses K/R as a catalytic base/acid pair [11].

Three-dimensional structures

Figure 2. Crystal structures of the monomeric chrondroitinase B and AlyF aswell as the dimeric AlyGC. Catalytic residues and substrates are in green the neutralizing calcium is the red sphere.

PL6 catalytic domain adopts a parallel β-helix fold with the activesite located on the surface of one of the β-sheets (Figure 2). The first PL6 structure solved was the chrondoitinase B from Pedobacter Heparinus (1.7 Å) [8] later it was shown that this enzyme is calcium dependent [9]. The first alginate lyase structure solved was the exolytic, guluronic acid-specific, homo-dimeric AlyGC in complex with tetra-mannuronic acid (2.6 Å) [6]. The first monomeric alginate lyase structure solved was the guluronic acid-specific AlyF in complex with tetra-guluronic acid (1.8 Å) [5]. All three structure belong to subfamily 1. There are no available crystal structures from subfamilies 2 and 3.

Family Firsts

First catalytic activity

OS-ALG-9 from Pseudomonas sp. on non-purified recombinant enzyme by the thiobarbituric acid method [7]

First catalytic base/acid

The catalytic arginine was originally identified in Chondroitinase B from Pedobacter Heparinus based on the crystal structure, concervation, mutagenesis and activity analysis (R271E no activity, R271K 0.09 % activity) [9]. The catalytic lysine was identified later by conservation, mutagenesis and activity analysis [6]

First charge neutralizer

Chondroitinase B from Pedobacter Heparinus by by crystallography and assaying the effect of calcium on enzyme activity [9].

First 3-D structure

Chondroitinase B from Pedobacter Heparinus [8].

References

1. 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 |
2. Mathieu S, Henrissat B, Labre F, Skjåk-Bræk G, and Helbert W. (2016). Functional Exploration of the Polysaccharide Lyase Family PL6. PLoS One. 2016;11(7):e0159415. DOI:10.1371/journal.pone.0159415 |

3. Haug, A., Larsen, B., and Smidsrod, O. (1966) A study of constitution of alginic acid by partial acid hydrolysis. Acta Chem. Scand. 20, 183–190

4. Haug, A., Larsen, B., and Smidsrod, O. (1967) Studies on sequence of uronic acid residues in alginic acid. Acta Chem. Scand. 21, 691–704

5. Lyu Q, Zhang K, Shi Y, Li W, Diao X, and Liu W. (2019). Structural insights into a novel Ca(2+)-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution. Biochim Biophys Acta Gen Subj. 2019;1863(7):1167-1176. DOI:10.1016/j.bbagen.2019.04.013 |
6. Xu F, Dong F, Wang P, Cao HY, Li CY, Li PY, Pang XH, Zhang YZ, and Chen XL. (2017). Novel Molecular Insights into the Catalytic Mechanism of Marine Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6. J Biol Chem. 2017;292(11):4457-4468. DOI:10.1074/jbc.M116.766030 |
7. Maki H, Mori A, Fujiyama K, Kinoshita S, and Yoshida T. (1993). Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp. OS-ALG-9. J Gen Microbiol. 1993;139(5):987-93. DOI:10.1099/00221287-139-5-987 |
8. Huang W, Matte A, Li Y, Kim YS, Linhardt RJ, Su H, and Cygler M. (1999). Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution. J Mol Biol. 1999;294(5):1257-69. DOI:10.1006/jmbi.1999.3292 |
9. Michel G, Pojasek K, Li Y, Sulea T, Linhardt RJ, Raman R, Prabhakar V, Sasisekharan R, and Cygler M. (2004). The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery. J Biol Chem. 2004;279(31):32882-96. DOI:10.1074/jbc.M403421200 |
10. Garron ML and Cygler M. (2010). Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology. 2010;20(12):1547-73. DOI:10.1093/glycob/cwq122 |
11. Xu F, Wang P, Zhang YZ, and Chen XL. (2018). Diversity of Three-Dimensional Structures and Catalytic Mechanisms of Alginate Lyases. Appl Environ Microbiol. 2018;84(3). DOI:10.1128/AEM.02040-17 |