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Polysaccharide Lyase Family 9

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Polysaccharide Lyase Family PL9
3D Structure β-helix
Mechanism β-elimination
Charge neutraliser calcium
Active site residues known
CAZy DB link
http://www.cazy.org/PL9.html

Substrate specificities

Polysaccharide lyases of family 9 (CAZy) are active on pectins, a major plant cell wall polysaccharide. The main activity in characterized PL9 is pectate lyase. These enzymes cleave non-methylated α-(1-4)-linked D-galacturonic acid (homogalacturonan) by a β-elimination mechanism (EC 4.2.2.2) [1]. Two PL9 endo-acting lyases have been shown to be active on rhamnogalacturonan-I (EC 4.2.2.23) [2]. Additional activities include: exopolygalacturonic lyase (EC 4.2.2.9) and thiopeptidoglycan lyase (EC 4.2.2.-) [3, 4].

Kinetics and Mechanism

PL9 acts by an anti-β-elimination mechanism generating a 4,5-unsaturated galacturonic acid product and a new reducing end. The elimination of C5 proton is base-catalyzed by lysine 237 [1]. Similar to the PL1 family, a calcium ion interacts with the substrate carboxylate at +1 subsite promoting the C5 proton acidification. [1, 5]. The characterization of the Bacteroides thetaiotaomicron rhamnogalacturonan lyase (BT4170) revealed an additional calcium ion also interacting with the substrate and playing a role in catalysis (Figure 1) [2].

Catalytic Residues

Figure 1. BT4170 (PDB ID 5OLR) and Pel9A (PDB ID 1RU4) active site. Superimposed active residues of BT4170 (cyan) and Pel9A (green). The calcium ions are represented as spheres (gray). The first calcium is found in both structures. However, Ca2+_2 is only present in BT4170 struture.

In Pel9A the lysine 237 (K237) is the Brønstead base (responsible for the abstraction of the C5 proton from galacturonic acid at +1 subsite). The calcium coordination pocket is comprised of four aspartates (D209, D233, D234 and D237) [1]. These residues are essential in catalysis and invariant in PL9 family (Figure 1) [2]. In BT4170 rhamnogalacturonan lyase, the residues G212, D246 and D280 comprise a second calcium binding site that is not conserved in pectate lyases (Figure 1) [2].

Three-dimensional structures

Figure 2. Pel9A in complex with Ca2+ (PDB ID 1RU4) A. Schematic representation of Pel9A parallel β-helix fold colour ramped from blue (N-terminal) to red (C-terminal). The active site is represented as sticks and highlighted inside the black box. The calcium is represented as sphere (gray) B. Blow up of the active site. The residues interacting with calcium and the proposed catalytic base (K237) are represented as stick in green and yellow, respectively.

PL9 structure of Erwinia chrysanthemi (Pel9A) was solved at a resolution of 1.6 Å (PDB ID 1RU4) and displays a right-handed parallel β-helix fold (Figure 2A). The superhelical structure presents 10 complete coils and 3 β -sheets (PB1, PB2, PB3). A short α-helix at N-terminus caps the hydrophobic core of the parallel β -helix. The catalytic base K237 and calcium binding site are orientated in the structure cleft (Figure 2B) [1]. The structure of the rhamnogalacturonan lyase (BT4170) in complex with the enzyme product showed that apart from the catalytic apparatus, there is little conservation of substrate binding residues between this enzyme and the pectate lyase Pel9A [2].

Family Firsts

First description of catalytic activity
PelX from Erwinia chrysanthemi [3].
First catalytic base identification
Pel9A from Erwinia chrysanthemi [1].
First catalytic divalent cation identification
Pel9A from Erwinia chrysanthemi [1].
First 3-D structure
Pel9A from Erwinia chrysanthemi [1].

References

  1. Jenkins J, Shevchik VE, Hugouvieux-Cotte-Pattat N, and Pickersgill RW. (2004) The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi. J Biol Chem. 279, 9139-45. DOI:10.1074/jbc.M311390200 | PubMed ID:14670977 | HubMed [Jenkins2004]
  2. Luis AS, Briggs J, Zhang X, Farnell B, Ndeh D, Labourel A, Baslé A, Cartmell A, Terrapon N, Stott K, Lowe EC, McLean R, Shearer K, Schückel J, Venditto I, Ralet MC, Henrissat B, Martens EC, Mosimann SC, Abbott DW, and Gilbert HJ. (2018) Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides. Nat Microbiol. 3, 210-219. DOI:10.1038/s41564-017-0079-1 | PubMed ID:29255254 | HubMed [Luis2018]
  3. Brooks AD, He SY, Gold S, Keen NT, Collmer A, and Hutcheson SW. (1990) Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product. J Bacteriol. 172, 6950-8. DOI:10.1128/jb.172.12.6950-6958.1990 | PubMed ID:2254266 | HubMed [Brooks1990]
  4. Kondo K, Takeda M, Ejima W, Kawasaki Y, Umezu T, Yamada M, Koizumi J, Mashima T, and Katahira M. (2011) Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide lyase, thiopeptidoglycan lyase. Int J Biol Macromol. 48, 256-62. DOI:10.1016/j.ijbiomac.2010.11.009 | PubMed ID:21095202 | HubMed [Kondo2011]
  5. Seyedarabi A, To TT, Ali S, Hussain S, Fries M, Madsen R, Clausen MH, Teixteira S, Brocklehurst K, and Pickersgill RW. (2010) Structural insights into substrate specificity and the anti beta-elimination mechanism of pectate lyase. Biochemistry. 49, 539-46. DOI:10.1021/bi901503g | PubMed ID:20000851 | HubMed [Seyedarabi2010]
All Medline abstracts: PubMed | HubMed