Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger

TY - JOUR

T1 - Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger

AU - Faulds, Craig B

AU - Molina, Rafael

AU - Gonzalez, Ramón

AU - Husband, Fiona

AU - Juge, Nathalie

AU - Sanz-Aparicio, Julia

AU - Hermoso, Juan A

PY - 2005/9

Y1 - 2005/9

N2 - 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.

AB - 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.

KW - Aspergillus niger/enzymology

KW - Base Sequence

KW - Carboxylic Ester Hydrolases/chemistry

KW - Catalytic Domain/genetics

KW - Crystallography, X-Ray

KW - DNA, Fungal/genetics

KW - Kinetics

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Phylogeny

KW - Pichia/enzymology

KW - Protein Conformation

KW - Recombinant Proteins/chemistry

KW - Substrate Specificity

U2 - 10.1111/j.1742-4658.2005.04849.x

DO - 10.1111/j.1742-4658.2005.04849.x

M3 - Journal article

C2 - 16128806

SN - 1742-4658

VL - 272

SP - 4362

EP - 4371

JO - The F E B S Journal (Online)

JF - The F E B S Journal (Online)

IS - 17

ER -