Polysaccharide Lyase Family 15

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• Author: ^^^Emil Stender^^^
• Responsible Curator: ^^^Birte Svensson^^^

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Polysaccharide Lyase Family 15
3D structure	(α/α)6 barrel + anti-parallel β-sheet
Mechanism	β-elimination
Charge neutralizer	Arginine and histidine
Active site residues	known
CAZy DB link
https://www.cazy.org/PL15.html

Substrate specificities

PL15 contains 2 subfamilies [1] as well as several proteins not assigned to any subfamily. Subfamily 1 has been shown to only degrade alginate [2, 3, 4, 5] while subfamily 2 has been found to be heparin and heparan sulfate lyases [6, 7]. 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 [8, 9]. Heparin consisting of disaccharide repeating units of which the most common is 2-O-sulfated 1,4 linked α-L-iduronic acid and 6-O-sulfated, N-sulfated glucosamine [IdoA(2S)-GlcNS(6S)]. Heparan sulfate being very similar to heparin having the IdoA replaced with β-D-glucuronic acid with a considerably more variable sulfation and acetylation pattern [10].

Kinetics and Mechanism

Figure 1 +1 subsite of the alginate lyase Atu3025

The β-elimination catalyzed by the PL15 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 the conserved H531 and R314 (Atu3025 numbering)[3]. This lowers the pKa 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.

Catalytic Residues

After charge neutralization a histidine functions as the catalytic base and a tyrosine the acid. They were originally identified as H311 and Y365 in Atu3025 from Agrobacterium fabrum [3].

Three-dimensional structures

Figure 2 Crystal structures of the substrate complex of the monomeric Atu3025 (PDB ID 3AFL) with the substrate in blue.

The first crystal structure available for a PL15 memberwas that of the alginate lyase Atu3025 from Agrobacterium fabrum (Figure 2) [3]. The catalytic domains consists of an N-terminal (α/α)₆ barrel domain and a C-terminal anti-parallel β-sheet domain. The catalytic site is located between the two domains with the catalytic residues and the arginine charge neutralizer located in the (α/α)₆ barrel and the histidine neutralizer in a loop extending into the active site from the anti-parallel β-sheet domain [3].

Family Firsts

First catalytic activity

Alginate lyase IV from Sphingomonas sp activity shown against alginate di- and trisaccharides by TLC from purified protein [2].

First catalytic base/acid

Atu3025 from Agrobacterium fabrum. H311 and Y365 was suggested as Acid/base based upon the crystal structure of the substrate complex, residue conservation, mutagenesis and activity analysis (H311A: inactive and Y365A: 0.3 % activity remaining)[3].

First charge neutralizer

Atu3025 from Agrobacterium fabrum H531 was suggested based on the crystal structure, its conservation, mutagenesis and activity analysis (H531A 0.45 % activity). R314 is proposed based on its proximity to the carboxylate group in the +1 subsite and its conservation [3].

First 3-D structure

Atu3025 from Agrobacterium fabrum [3].

References

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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 |

8. Haug, A., Larsen, B., and Smidsrod, O. (1967) Studies on sequence of uronic acid residues in alginic acid. Acta Chem. Scand. 21, 691–704. DOI:10.3891/acta.chem.scand.21-0691

9. 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. DOI:10.3891/acta.chem.scand.20-0183

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 |