Polysaccharide Lyase Family 4

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• Author: ^^^Leila LoLeggio^^^ and Sine Larsen
• Responsible Curator: ^^^Leila LoLeggio^^^

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Polysaccharide Lyase Family PL4
Mechanism	β-elimination
Charge neutraliser	none
Active site residues	known
CAZy DB link
https://www.cazy.org/PL4.html

Substrate specificities

The main activity assigned to characterized enzymes in PL4 is degradation of the plant cell wall component rhamnogalacturonan I, a component of pectin hairy regions. Rhamnogalacturonan I is a heteropolymer built up by the disaccharide unit [α-L-Rha-(1,4)-α-d-GalUA-(1,2)], with often extensive branching (arabinans, galactans and arabinogalactans)at the O2 and O3 of the galacturonic acid units. Both rhamnose and galacturonic acid units are present in Rhamnogalacturonan I in their pyranose forms. Characterized PL4 enzymes are therefore Rhamnogalacturonan lyases (EC 4.2.2.23) The best characterized enzyme in the family, the Aspergillus aculeatus Rhamnogalacturonan Lyase (AaRGL4) [1] cleaves the α-1,4-glycosidic bonds between L-rhamnose and D-galacturonic acids, and produces an unsaturated product with α-Δ-(4,5)- d-galacturonic acid at the non-reducing end [2]. Other biochemical studies [3] showed that the minimum substrate requirement is a deacetylated dodecamer, with preferential cleavage four residues from the reducing end Rha, but the structural studies (see below) have demonstrated that smaller ligands can be bound [4]. The effect of branching depends on the nature of the side chains, as removal of arabinan chains increases activity, while removal of galactose side chains reduces activity [3]. The branching effects may account for the fact that some experiments have shown an average size of 25-30 sugar units in complete digestions of rhamnogalacturonan I by AaRGL4 [4].In CAZY [5, 6], PL4 is divided in 5 subfamilies with members from bacterial and eukaryotic kingdoms (fungi and plants). Apart from subfamily 2, consisting primarily of plant members, the subfamilies do not seem to follow phylogenetic divisions, and may reflect yet undiscovered differences in substrate preferences.

Kinetics and Mechanism

Degradation of rhamnogalacturonan is via β-elimination, which introduces a double bond in the non-reducing D-gactopyranosyluronic acid unit. The optimum pH of activity is low (pH 6.00 as reported for AaRGL4 [3]) compared to other polysaccharide lyases, which tend to have rather basic pH optima. Another major difference to other polysaccharide lyase mechanisms, is that divalent metal ions are not required by PL4 for catalysis. Both the low pH optimum and the lack of strict metal requirement show parallels between the PL4 mechanism and the mechanism of pectin lyases []. A mechanism for PL4 has been proposed based on mutagenesis and structural studies in complex with a reaction product (see below)[4].

Catalytic Residues

Catalytic residues were first suggested on the basis of sequence conservation and location on the 3D structure [7], and subsequently verified by site directed mutagenesis in AaRGL4 to be Lys150 and His210[4]. In the proposed mechanism [4], based both on mutagenesis and structural considerations, Lys150 is the proton abstractor, while His210 plays the role of proton donor. In most other polysaccharide lyase mechanisms a proton donor has not been identified. A Lys as proton abstractor seems in conflict with the low pH optimum, but pKa calculations with a model of a substrate complex suggest that desolvation effects may help lower the pKa of this residue [4].

Three-dimensional structures

Content is to be added here.

Family Firsts

First demonstration of unsaturated product

Aspergillus aculeatus Rhamnogalacturonan lyase [2].

First proton abstractor identification

Aspergillus aculeatus Rhamnogalacturonan lyase [4].

First proton donor identification

Aspergillus aculeatus Rhamnogalacturonan lyase [4].

First 3-D structure

Aspergillus aculeatus Rhamnogalacturonan lyase [7].

References

1. Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, Dörreich K, and Dalbøge H. (1994). Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. J Biol Chem. 1994;269(46):29182-9. | Google Books | Open Library
2. Azadi P, O'Neill MA, Bergmann C, Darvill AG, and Albersheim P. (1995). The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase. Glycobiology. 1995;5(8):783-9. DOI:10.1093/glycob/5.8.783 |
3. Mutter M, Colquhoun IJ, Beldman G, Schols HA, Bakx EJ, and Voragen AG. (1998). Characterization of recombinant rhamnogalacturonan alpha-L-rhamnopyranosyl-(1,4)-alpha-D-galactopyranosyluronide lyase from Aspergillus aculeatus. An enzyme that fragments rhamnogalacturonan I regions of pectin. Plant Physiol. 1998;117(1):141-52. DOI:10.1104/pp.117.1.141 |
4. Jensen MH, Otten H, Christensen U, Borchert TV, Christensen LL, Larsen S, and Leggio LL. (2010). Structural and biochemical studies elucidate the mechanism of rhamnogalacturonan lyase from Aspergillus aculeatus. J Mol Biol. 2010;404(1):100-11. DOI:10.1016/j.jmb.2010.09.013 |

5. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. Biochem. J. (BJ Classic Paper, online only). DOI: 10.1042/BJ20080382

6. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |
7. McDonough MA, Kadirvelraj R, Harris P, Poulsen JC, and Larsen S. (2004). Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4. FEBS Lett. 2004;565(1-3):188-94. DOI:10.1016/j.febslet.2004.03.094 |