PDBsum entry 2bjh

Hydrolase

PDB id: 2bjh

Contents

Protein chains

260 a.a. *

Ligands

NAG ×3

NDG ×3

FER ×3

Waters ×373

* Residue conservation analysis

PDB id:

2bjh

Name:

Hydrolase

Title:

Crystal structure of s133a anfaea-ferulic acid complex

Structure:

Feruloyl esterase a. Chain: a, b, c. Synonym: ferulic acid esterase a, fae-iii, cinnamoyl esterase. Engineered: yes. Mutation: yes

Source:

Aspergillus niger. Organism_taxid: 5061. Strain: cbs 120.49/n400. Expressed in: pichia pastoris. Expression_system_taxid: 4922

Resolution:

2.54Å R-factor: 0.216 R-free: 0.277

Authors:

C.B.Faulds,R.Molina,R.Gonzalez,F.Husband,N.Juge,J.Sanz-Aparicio, J.A.Hermoso

Key ref:

C.B.Faulds et al. (2005). Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger. FEBS J, 272, 4362-4371. PubMed id: 16128806 DOI: 10.1111/j.1742-4658.2005.04849.x

Date:

02-Feb-05 Release date: 07-Sep-05

Protein chains

O42807  (FAEA_ASPNG) -  Feruloyl esterase A from Aspergillus niger

Seq: 281 a.a.

Struc: 260 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.1.73 - feruloyl esterase.
• Reaction: feruloyl-polysaccharide + H2O = ferulate + polysaccharide

feruloyl-polysaccharide + H2O = ferulate (Bound ligand (Het Group name = FER) corresponds exactly) + polysaccharide

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1111/j.1742-4658.2005.04849.x

FEBS J 272:4362-4371 (2005)

PubMed id: 16128806

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

C.B.Faulds, R.Molina, R.Gonzalez, F.Husband, N.Juge, J.Sanz-Aparicio, J.A.Hermoso.

ABSTRACT

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.

Selected figure(s)

Figure 2.
Fig. 2. Crystal structure of the S133A AnFaeA mutant in complex with FAXX. (A) Molecular surface of S133A AnFaeA mutant. The catalytic triad and the Y80 and W260 residues are labelled. Ferulic acid molecule is coloured in cyan. (B) Environment of FA (green) in the active site of S133A. The flap region of AnFaeA is highlighted in dark blue. (C) Proposed interactions of FA with residues at the substrate cavity of AnFaeA.

Figure 4.
Fig. 4. Local environment of Tyr80 and Trp260. (A) Arrangement of the Tyr80 residue. Ferulic acid is shown in blue, Tyr80 is shown in red and the residues that participate in the substrate cavity are shown in green. (B) Arrangement of Trp260 residue (red). The residues that bury Tpr260 are shown in blue.

The above figures are reprinted by permission from the Federation of European Biochemical Societies: FEBS J (2005, 272, 4362-4371) copyright 2005.

Figures were selected by an automated process.