CAZypedia needs your help!
We have many unassigned pages in need of Authors and Responsible Curators. See a page that's out-of-date and just needs a touch-up? - You are also welcome to become a CAZypedian. Here's how.
Scientists at all career stages, including students, are welcome to contribute.
Learn more about CAZypedia's misson here and in this article.
Totally new to the CAZy classification? Read this first.

Polysaccharide Lyase Family 4

From CAZypedia
(Redirected from PL4)
Jump to navigation Jump to search
Approve icon-50px.png

This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.

Polysaccharide Lyase Family PL4
Mechanism β-elimination
Charge neutraliser none
Active site residues known
CAZy DB link

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 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) [7]. 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 such as pectate lyases, which tend to have rather basic pH optima. Another major difference to the pectate and 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 [8], which despite belonging to PL1, have diverged significantly from the pectate lyases. A mechanism for PL4 has been proposed based on mutagenesis and structural studies of a ligand complex (see below)[4].

Catalytic Residues

Catalytic residues were first suggested on the basis of sequence conservation and location on the 3D structure [9], 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

Structure of AaRGL4 K150A in complex with a hexasaccharide. Domain I is in magenta, domain II in cyan, and domain III in blue. The product is shown in sphere representation. Figure made in pymol

The three-dimensional structure of PL4 rhamnogalacturonan lyase from Aspergillus aculeatus AaRGL4 [9] [1NKG], revealed that it is shaped as a flattened oval disk of approximate dimensions 90 x 58 x 40Å. The secondary structure is predominantly β-sheet arranged into three distinct modular domains, I, II and III. The N-terminal domain I, containing the catalytic residues and formed by residues 1-257, is folded into a β-super-sandwich, the fold of domain II comprised by residues 258-336 has a topology that is similar to fibronectin type III (FnIII). The residues 337-508 form the C-terminal domain III which displays a jelly roll β-sandwich fold and is structurally homologous to carbohydrate binding modules. The C-terminal hosts a structural calcium ion, not thought to be involved in catalysis. The structures of catalytic residues variants [2XHN, 3NJX]and a complex of the K150A catalytically impaired variant with substrate bound from -3 to +3 subsites[3NJV]have been also determined [4].

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 [9].


  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 PubMed ID:7961884 [Kofod1994]
  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 | PubMed ID:8720076 [Azadi1995]
  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 | PubMed ID:9576783 [Mutter1998]
  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 | PubMed ID:20851126 [Jensen2010]
  5. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. The Biochemist, vol. 30, no. 4., pp. 26-32. Download PDF version.

  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 | PubMed ID:18838391 [Cantarel2009]
  7. 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 | PubMed ID:20925655 [Lombard2010]
  8. Mayans O, Scott M, Connerton I, Gravesen T, Benen J, Visser J, Pickersgill R, and Jenkins J. (1997). Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases. Structure. 1997;5(5):677-89. DOI:10.1016/s0969-2126(97)00222-0 | PubMed ID:9195887 [Mayans1997]
  9. 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 | PubMed ID:15135077 [McDonough2004]

All Medline abstracts: PubMed