The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition.
BACKGROUND
Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications.


RESULTS
We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum. The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0%.


CONCLUSIONS
The C. thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.
Prates
J A
JA
Centro Interdisciplinar de Investigação em Sanidade Animal, Faculdade de Medicina Veteterinária, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa Codex, Portugal.
Tarbouriech
N
N
Charnock
S J
SJ
Fontes
C M
CM
Ferreira
L M
LM
Davies
G J
GJ
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D004583:Electrons; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012645:Selenomethionine / Q000737:chemistry; D013379:Substrate Specificity; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics
D012645:Selenomethionine; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase