Structural Basis for the Substrate Specificity of the Feruloyl Esterase Domain of the Cellulosomal Xylanase Z from Clostridium thermocellum

Abstract

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

“…5A). Amino acid alignments also revealed that a catalytic triad (Ser-His-Glu) that is crucial for catalysis in ferulic acid esterase enzymes (6,58) is present and that S629 may be the catalytic nucleophile for the esterase domain of Xyn10D-Fae1A (Fig. 5A).…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…Polysaccharide lyases cleave their scissile bond through a b-elimination mechanism (Herron et al, 2000). While carbohydrate esterases generally hydrolyze ester linkages through a double displacement mechanism in which Ser (Schubot et al, 2001) or Asp in CE8 (Fries et al, 2007) functions as the catalytic nucleophile, exceptions to this mode of action are apparent in CE4, where catalysis is metal dependent .…”

The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases

“…Analysis of the available FAE sequences and available crystal structures by several researches shows that the active site of FAEs contains the catalytic triad (Ser, H is, Asp), and the serine residue is located at the centre of universally conserved pentapeptide with the consensus 'nucleophilic elbow' i.e., GXSXG (X=any amino acid residue) (Schubot et al, 2001;Prates et al, 2001;T arbouriech et al, 2005;H ermoso et al, 2004;McAuley et al, 2004;Faulds et al, 2005;Benoit et al, 2006b). So, the presence of the catalytic triad with the serine containing nucleophilic elbow in a particular candidate sequence denotes a high probability for it being a putative FAE.…”