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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) degrade homogalacturonan,a pectin component present in the plant cell walls. An enzyme in PL9 was described as active on sheath, a thioloic glycoconjugate secreted by Sphaerotilus natans [1]. The main activity in characterized PL9 is pectate lyases. These enzymes cleave non-methylated α-(1-4)-linked D-galacturonic acid by a β-elimination mechanism (EC 4.2.2.2) [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 [2]. Similar to the PL1 family, a calcium ion interacts with the substrate carboxylate at +1 subsite promoting the C5 proton acidification. [2, 5].

Catalytic Residues

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) [2].

Three-dimensional structures

Figure 1. 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 1A). 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 1B) [2].

Family Firsts

First description of catalytic activity
PelX from Erwinia chrysanthemi [3].
First catalytic base identification
Pel9A from Erwinia chrysanthemi [2].
First catalytic divalent cation identification
Pel9A from Erwinia chrysanthemi [2].
First 3-D structure
Pel9A from Erwinia chrysanthemi [2].

References

  1. Takeda M, Iohara K, Shinmaru S, Suzuki I, and Koizumi JI. (2000) Purification and properties of an enzyme capable of degrading the sheath of Sphaerotilus natans. Appl Environ Microbiol. 66, 4998-5004. PubMed ID:11055955 | HubMed [Takeda2000]
  2. 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]
  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. 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