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The endo-polygalacturonase II (Polygalacturonase EC 3.2.1.15) of the fungus ''Aspergillus Niger'' is an enzyme which is involved in the rotting process. It specifically degrade polygalacturonate, a major carbohydrate constituent of plant cell wall pectin, by hydrolysis of the glycosidic bonds that link galacturonic acid residues. | The endo-polygalacturonase II (Polygalacturonase EC 3.2.1.15) of the fungus ''Aspergillus Niger'' is an enzyme which is involved in the rotting process. It specifically degrade polygalacturonate, a major carbohydrate constituent of plant cell wall pectin, by hydrolysis of the glycosidic bonds that link galacturonic acid residues. | ||
Polygalacturonase belongs to the Glycoside hydrolysases 28 (GH28) according to the sequence-based classification of glycoside hydrolysases. The enzyme of the GH28 family achieves the hydrolysis with an inverting molecular mechanism. | Polygalacturonase belongs to the Glycoside hydrolysases 28 (GH28) according to the sequence-based classification of glycoside hydrolysases. The enzyme of the GH28 family achieves the hydrolysis with an inverting molecular mechanism.<ref>http://www.cazypedia.org/index.php/Inverting#Mechanistic_classification</ref> | ||
== Glycoside hydrolases and pectinases == | == Glycoside hydrolases and pectinases == | ||
The endopolygalacturonase II belongs to the family of glycoside hydrolases, enzymes that hydrolyse glycosidic bonds in polysaccharidic chains and release smaller sugars, classified in the EC 3.2.1.x group. Within this family, endopolygalacturonases are pectinases, enzymes involved in the degradation of pectin, a polymer of galacturonic acid and rhamnose that is the jelly-like component of plant cell walls. | |||
The endopolygalacturonase II belongs to the family of glycoside hydrolases, enzymes that hydrolyse glycosidic bonds in polysaccharidic chains and release smaller sugars, classified in the EC 3.2.1.x group. Within this family, endopolygalacturonases are pectinases, enzymes involved in the degradation of pectin, a polymer of galacturonic acid and rhamnose that is the jelly-like component of plant cell walls. | |||
Endopolygalacturonase II is part of the family 28 of glycoside hydrolases. It is not the only endopolygalacturonase from A. niger - a family of seven genes encode such enzymes, and six different proteins have been identified: endopolygalacturonases I, II, A, B, C and E. | Endopolygalacturonase II is part of the family 28 of glycoside hydrolases. It is not the only endopolygalacturonase from A. niger - a family of seven genes encode such enzymes, and six different proteins have been identified: endopolygalacturonases I, II, A, B, C and E. | ||
== The catalysed reaction == | == The catalysed reaction == | ||
The endopolygalacturonase II is an endo-acting enzyme that hydrolyses alpha(1,4)-glycosidic bonds between the D-galacturonic acid monomeres of pectin chains. One of its particularities is the inversion of configuration that occurs during the reaction. Its functionning is optimal at a pH of 4.2 and with a non-methylated substrate. | The endopolygalacturonase II is an endo-acting enzyme that hydrolyses alpha(1,4)-glycosidic bonds between the D-galacturonic acid monomeres of pectin chains. One of its particularities is the inversion of configuration that occurs during the reaction. Its functionning is optimal at a pH of 4.2 and with a non-methylated substrate.<ref>http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.1999.00080.x/full</ref> | ||
[[Image:mechanisme.png |center]] | |||
The putative mechanism is the following: | The putative mechanism is the following: | ||
Asp180 and Asp202 bind the water molecule used in the hydrolyse and activate it, while Asp201, which is less exposed to the aqueous solvent, is stabilized its protonated state. In theses conditions, a bond can be built between the oxygen of the water and the carbon 1 of the galacturonic acid, whereas the bond between this carbon and the oxygen of the glycosidic bond is cleaved, the oxygen taking the proton released by Asp201. | Asp180 and Asp202 bind the water molecule used in the hydrolyse and activate it, while Asp201, which is less exposed to the aqueous solvent, is stabilized its protonated state. In theses conditions, a bond can be built between the oxygen of the water and the carbon 1 of the galacturonic acid, whereas the bond between this carbon and the oxygen of the glycosidic bond is cleaved, the oxygen taking the proton released by Asp201.<ref>http://www.jbc.org/content/275/38/29348.short</ref> | ||
[[Image:proposed mechanism.png | 332 px |center|thumb|'''Proposed mechanism for family 28 of glycoside hydrolases''']] | |||
== Description of the structure == | == Description of the structure == | ||
{{STRUCTURE_1czf| PDB=1czf | SCENE= }} | |||
The loops separating PB1 and PB2a are longer on the C-terminal end, and those between PB3 and PB1 on the N-terminal end, what forms a cleft between two extensions outside of the the beta-helix. Its shape, open at both ends of the protein, allows the fixation of a linear glucidic chain, and is suited to the endohydrolytic mode of action. This cleft is a higly conserved region. | The endopolygalacturonase II from Aspergillus niger has a beta-helix structure<ref>http://www.ncbi.nlm.nih.gov/pubmed/10521427</ref>: the chain folds into 10 turns, shaping the faces of a right-handed helix consisting in 4 parallel beta-sheets - PB1, PB2a, PB2b and PB3 - separated by loops. The helix is closed at its N-terminal end by a <scene name='Sandbox_Reserved_711/N_terminal_alpha_helix/1'>small alpha-helix</scene>. | ||
4 disulfide bridges, conserved in all endopolygalacturonases of ''A. Niger'', hold together different parts of the chain, and particularily one of these attaches the N-terminal helix to the PB2b sheet. | |||
The <scene name='Sandbox_Reserved_711/Chain_b_cleft/1'>loops separating PB1 and PB2a</scene> are longer on the C-terminal end, and those between PB3 and PB1 on the N-terminal end, what forms a cleft between two extensions outside of the the beta-helix. Its shape, open at both ends of the protein, allows the fixation of a linear glucidic chain, and is suited to the endohydrolytic mode of action. This cleft is a higly conserved region. | |||
8 particular amino-acids in this region are strictly conserved among polygalacturonases from other fungal and bacterial species: Asn178, Asp180, Asp201, Asp202, His223, Gly224, Arg256, and Lys258. | <scene name='Sandbox_Reserved_711/Active_site_residues/1'>8 particular amino-acids</scene> in this region are strictly conserved among polygalacturonases from other fungal and bacterial species: Asn178, Asp180, Asp201, Asp202, His223, Gly224, Arg256, and Lys258 (shown in red). | ||
The residues directly involved in the catalytic activity seem to be Asp180, Asp201, Asp202, and His223, that form a plane above which space is available, while Arg256 and Lys258 would bind the substrate. Gly224, at the bottom of the cleft, doesn't point out of the helix. | The residues directly involved in the catalytic activity seem to be <scene name='Sandbox_Reserved_711/Active_site_residues/3'>Asp180, Asp201, Asp202, and His223</scene> (orange), that form a plane above which space is available, while <scene name='Sandbox_Reserved_711/Active_site_residues/4'>Arg256 and Lys258 </scene> (light pink) would bind the substrate.<scene name='Sandbox_Reserved_711/Active_site_residues/5'>Gly224</scene> (dark pink), at the bottom of the cleft, doesn't point out of the helix. | ||
== Endo-polygalacturonase II applications == | == Endo-polygalacturonase II applications == | ||
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== References == | == References == | ||
<references/> | |||
== External links == | == External links == | ||
PDB entry: 1CZF[http://www.rcsb.org/pdb/explore.do?structureId=1czf] | |||
== Contributors == | == Contributors == | ||
Claire Baranger, Adeline Pauli | Claire Baranger, Adeline Pauli | ||