 |
PDBsum entry 2bjh
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Probing the determinants of substrate specificity of a feruloyl esterase, Anfaea, From aspergillus niger.
|
|
|
Authors
|
|
C.B.Faulds,
R.Molina,
R.Gonzalez,
F.Husband,
N.Juge,
J.Sanz-Aparicio,
J.A.Hermoso.
|
|
|
Ref.
|
|
FEBS J, 2005,
272,
4362-4371.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
