Pectate lyase L (polysaccharide lyase 9 family)

 

Endo-acting pectate lyase L (Pel9A) is from the bacteria Erwini chrysanthemi. It catalyses the eliminative cleavage of pectate to give oligosaccharides with 4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends. This particular bacteria causes soft rot diseases. It secretes enzymes such as Pel9A which break down pectin, a key component of cell walls.

 

Reference Protein and Structure

Sequence
P0C1A7 UniProt (4.2.2.2) IPR012334 (Sequence Homologues) (PDB Homologues)
Biological species
Dickeya dadantii 3937 (Bacteria) Uniprot
PDB
1ru4 - Crystal structure of pectate lyase Pel9A (1.6 Å) PDBe PDBsum 1ru4
Catalytic CATH Domains
2.160.20.10 CATHdb (see all for 1ru4)
Cofactors
Calcium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.2.2.2)

oligogalacturonide
CHEBI:62533ChEBI
→
4-(4-deoxy-alpha-D-gluc-4-enosyluronic acid)-D-galacturonic acid
CHEBI:27450ChEBI
+
alpha-D-galacturonic acid
CHEBI:33885ChEBI
Alternative enzyme names: Alpha-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N, Polygalacturonic acid trans-eliminase, Endo-alpha-1,4-polygalacturonic acid lyase, Endogalacturonate transeliminase, Endopectin methyltranseliminase, Pectate transeliminase, Pectic acid lyase, Pectic acid transeliminase, Pectic lyase, Pectin trans-eliminase, Polygalacturonate lyase, Polygalacturonic acid lyase, Polygalacturonic transeliminase,

Enzyme Mechanism

Introduction

The presence of a putative catalytic base but no clear catalytic acid suggests that the favoured reaction pathway would be E1cb. This is further substantiated by the Calcium ion interaction with the substrate carboxylate at C5. In this mechanism Ca(II) and Asn interact with the substrate carboxylate, making the C5 proton more acidic. Then, Lys273 acts as a base by taking the C5 proton, forming the substrate's conjugate base. The carboxylate reforms and the C4-O bond is cleaved. Solvent water protonates the glycosidic oxygen as it leaves.

Catalytic Residues Roles

UniProt PDB* (1ru4)
Lys273 Lys273(248)A Lys 273 deprotonates atom C5 of the substrate, to form the conjugate base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asn268 Asn268(243)A Activates the substrate by acidifying the C5 proton. It stabilises the conjugate base. hydrogen bond donor, electrostatic stabiliser, increase acidity
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

assisted keto-enol tautomerisation, proton transfer, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, intermediate collapse, intermediate terminated, overall product formed, native state of enzyme regenerated, inferred reaction step

References

  1. Jenkins J et al. (2004), J Biol Chem, 279, 9139-9145. The Crystal Structure of Pectate Lyase Pel9A from Erwinia chrysanthemi. DOI:10.1074/jbc.m311390200. PMID:14670977.

Step 1. Lys273 deprotonates the C5 of the poly(1,4-alpha-D-galacturonate) substrate. The C5 proton is acidified by the interaction of the substrate carboxylate with the calcium ion, the NH2 group of Asn268 and the resonant nature of the intermediate [PMID:14670977].

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Catalytic Residues Roles

Residue Roles
Lys273(248)A hydrogen bond acceptor
Asn268(243)A hydrogen bond donor, electrostatic stabiliser
Asn268(243)A increase acidity
Lys273(248)A proton acceptor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, overall reactant used, intermediate formation

Step 2. The oxyanion initiates an elimination reaction that releases the products, and the poly(1,4-alpha-D-galacturonate) deprotonates an acid. As there is no obvious enzyme or substrate Bronsted acid to protonate the glycosidic oxygen in this enzyme, protonation is presumed to occur via solvent water [PMID:14670977].

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Catalytic Residues Roles

Residue Roles
Asn268(243)A hydrogen bond donor, electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, intermediate terminated, overall product formed

Step 3. Water deprotonates Lys273.

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Catalytic Residues Roles

Residue Roles
Lys273(248)A hydrogen bond donor, proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Lys273 deprotonates the C5 of the poly(1,4-alpha-D-galacturonate) substrate. The C5 proton is acidified by the interaction of the substrate carboxylate with the calcium ion, the NH2 group of Asn268 and the resonant nature of the intermediate [PMID:14670977].

Step 2. The oxyanion initiates an elimination reaction that releases the products, and the poly(1,4-alpha-D-galacturonate) deprotonates an acid. As there is no obvious enzyme or substrate Bronsted acid to protonate the glycosidic oxygen in this enzyme, protonation is presumed to occur via solvent water [PMID:14670977].

Step 3. Water deprotonates Lys273.

Products.

Introduction

E1 proposal in which the substrate undergoes heterolysis followed by proton transfer to generate the final products.

Catalytic Residues Roles

UniProt PDB* (1ru4)
Lys273 Lys273(248)A Helps stabilise the charged intermediates and transition states formed during the course of the reaction. hydrogen bond acceptor, electrostatic stabiliser
Asn268 Asn268(243)A Helps stabilise the charged intermediates and transition states formed during the course of the reaction. Also acidifies the C5 proton. hydrogen bond donor, electrostatic stabiliser, increase acidity
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

overall reactant used, intermediate formation, heterolysis, proton transfer, overall product formed, native state of enzyme regenerated

References

  1. Jenkins J et al. (2004), J Biol Chem, 279, 9139-9145. The Crystal Structure of Pectate Lyase Pel9A from Erwinia chrysanthemi. DOI:10.1074/jbc.m311390200. PMID:14670977.

Step 1. The substrate C6-O bond undergoes heterolysis.

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Catalytic Residues Roles

Residue Roles
Lys273(248)A hydrogen bond acceptor
Asn268(243)A hydrogen bond donor, electrostatic stabiliser
Lys273(248)A electrostatic stabiliser

Chemical Components

overall reactant used, intermediate formation, heterolysis

Step 2. The negatively charged intermediate then abstracts the C5 proton, which is acidified by the interaction of the substrate carboxylate with the calcium ion, the NH2 group of Asn268 and the resonant nature of the intermediate [PMID:14670977]. This forms the final products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn268(243)A electrostatic stabiliser
Lys273(248)A electrostatic stabiliser
Asn268(243)A increase acidity

Chemical Components

proton transfer, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The substrate C6-O bond undergoes heterolysis.

Step 2. The negatively charged intermediate then abstracts the C5 proton, which is acidified by the interaction of the substrate carboxylate with the calcium ion, the NH2 group of Asn268 and the resonant nature of the intermediate [PMID:14670977]. This forms the final products.

Products.

Introduction

E2 proposal in which Lys273 abstracts the C5 proton from the substrate, initiating the elimination of the alcohol product with concomitant abstraction of a proton from a general acid (probably water).

Catalytic Residues Roles

UniProt PDB* (1ru4)
Lys273 Lys273(248)A Lys 273 deprotonates atom C5 of the substrate, to form the conjugate base. hydrogen bond acceptor, proton acceptor, proton donor
Asn268 Asn268(243)A Also acidifies the C5 proton. hydrogen bond donor, electrostatic stabiliser, increase acidity
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, overall reactant used, bimolecular elimination, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Jenkins J et al. (2004), J Biol Chem, 279, 9139-9145. The Crystal Structure of Pectate Lyase Pel9A from Erwinia chrysanthemi. DOI:10.1074/jbc.m311390200. PMID:14670977.

Step 1. Lys273 deprotonates the C5 of the poly(1,4-alpha-D-galacturonate) substrate. The C5 proton is acidified by the interaction of the substrate carboxylate with the calcium ion, the NH2 group of Asn268 and the resonant nature of the intermediate [PMID:14670977]. This initiates the elimination of the alcohol product with concomitant deprotonation of an acid, assumed to be a water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys273(248)A hydrogen bond acceptor
Asn268(243)A hydrogen bond donor, electrostatic stabiliser, increase acidity
Lys273(248)A proton acceptor

Chemical Components

proton transfer, overall reactant used, ingold: bimolecular elimination, overall product formed

Step 2. Inferred return step in which a water/hydroxide abstracts the proton from Lys273.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys273(248)A proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Lys273 deprotonates the C5 of the poly(1,4-alpha-D-galacturonate) substrate. The C5 proton is acidified by the interaction of the substrate carboxylate with the calcium ion, the NH2 group of Asn268 and the resonant nature of the intermediate [PMID:14670977]. This initiates the elimination of the alcohol product with concomitant deprotonation of an acid, assumed to be a water molecule.

Step 2. Inferred return step in which a water/hydroxide abstracts the proton from Lys273.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Ellie Wright