2bjh: Difference between revisions
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==Overview== | ==Overview== | ||
Feruloyl esterases hydrolyse phenolic groups involved in the cross-linking, of arabinoxylan to other polymeric structures. This is important for, opening the cell wall structure making material more accessible to, glycoside hydrolases. Here we describe the crystal structure of inactive, S133A mutant of type-A feruloyl esterase from Aspergillus niger (AnFaeA), in complex with a feruloylated trisaccharide substrate. Only the ferulic, acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH(3) groups with the hydroxyl, groups of Tyr80. The importance of aromatic and polar residues in the, activity of AnFaeA was also evaluated using site-directed mutagenesis., Four mutant proteins were heterologously expressed in Pichia pastoris, and, ... | Feruloyl esterases hydrolyse phenolic groups involved in the cross-linking, of arabinoxylan to other polymeric structures. This is important for, opening the cell wall structure making material more accessible to, glycoside hydrolases. Here we describe the crystal structure of inactive, S133A mutant of type-A feruloyl esterase from Aspergillus niger (AnFaeA), in complex with a feruloylated trisaccharide substrate. Only the ferulic, acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH(3) groups with the hydroxyl, groups of Tyr80. The importance of aromatic and polar residues in the, activity of AnFaeA was also evaluated using site-directed mutagenesis., Four mutant proteins were heterologously expressed in Pichia pastoris, and, their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid. The k(cat) of Y80S, Y80V, W260S and, W260V was drastically reduced compared to that of the wild-type enzyme., However, the replacement of Tyr80 and Trp260 with smaller residues, broadened the substrate specificity of the enzyme, allowing the hydrolysis, of methyl caffeate. The role of Tyr80 and Trp260 in AnFaeA are discussed, in light of the three-dimensional structure. | ||
==About this Structure== | ==About this Structure== | ||
2BJH is a | 2BJH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aspergillus_niger Aspergillus niger] with NAG, NDG and FER as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Feruloyl_esterase Feruloyl esterase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.73 3.1.1.73] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2BJH OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: xylan degradation]] | [[Category: xylan degradation]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 14:32:08 2007'' | ||
