Crystal structure and snapshots along the reaction pathway of a family 51  -L-arabinofuranosidase

Abstract

contributed equally to this work High-resolution crystal structures of a-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycosidase, are described. The enzyme is a hexamer, and each monomer is organized into two domains: a (b/a) 8 -barrel and a 12-stranded b sandwich with jelly-roll topology. The structures of the Michaelis complexes with natural and synthetic substrates, and of the transient covalent arabinofuranosyl± enzyme intermediate represent two stable states in the double displacement mechanism, and allow thorough examination of the catalytic mechanism. The arabinofuranose sugar is tightly bound and distorted by an extensive network of hydrogen bonds. The two catalytic residues are 4.7 A Ê apart, and together with other conserved residues contribute to the stabilization of the oxocarbenium ion-like transition state via charge delocalization and speci®c protein±substrate interactions. The enzyme is an anti-protonator, and a 1.7 A Ê electrophilic migration of the anomeric carbon takes place during the hydrolysis. Keywords: arabinofuranosidase/clan GH-A glycosidase/ enzyme mechanism/glycoside hydrolase family 51/X-ray crystallography IntroductionMost of the biomass synthesized by photosynthetic CO 2 ®xation is stored in the plant cell wall as polymeric carbohydrates, mainly in the form of cellulose and hemicellulose. The degradation of these polymers is a key step in the carbon cycle, and is mediated by microorganisms that produce speci®c enzymes, i.e. cellulases and hemicellulases ( Figure 1A) . a-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that hydrolyze the arabinofuranosyl substitutions in hemicellulose. Some of these enzymes exhibit broad substrate speci®city, acting on arabinofuranoside moieties at O-2 or O-3 as a single substituent, as well as from O-2 and O-3 doubly substituted xylans, xylooligomers and a-1,5-linked arabinans (Saha, 2000). The glycosidic bond between two sugars is one of the most stable bonds in nature, and its enzymatic hydrolysis, carried out by glycoside hydrolases, provides an acceleration rate (k cat /k uncat ) which can be as high as 10 17 -fold (Wolfenden et al., 1998). The key elements in this remarkable catalysis are the ®nely tuned positions of the catalytic residues, and the distortion of the sugar ring so as to allow the stabilization of an oxocarbenium-ion-like transition state, as well as the movement of the hydrogen on the anomeric carbon to allow a direct nucleophilic attack (Zechel and Withers, 1999). Many glycosidases are modular proteins, and in addition to their catalytic domains include other functional modules, mainly carbohydrate-binding modules (CBMs) (Bourne and Henrissat, 2001). Currently, >8500 glycosidase sequences are known, and the sequence-based classi®cation of their catalytic domains into glycoside hydrolase (GH) families and clans is available on the continuously updated Carbohydrate-Active Enzymes (CAZy) server (http:// afmb.cnrs-mrs.fr/CAZY). The different bacterial, fungal and plant a-L-arabinofuranosidases...

Crystal Structure of a Family 54 α-l-Arabinofuranosidase Reveals a Novel Carbohydrate-binding Module That Can Bind Arabinose

“…If so, the conformation is the same as that of the Michaelis complex with arabinofuranose-␣ (1, 3)-xylopyranose reported for GH51 GsAbfA (Fig. 5B) (15). However, the limited crystallographic resolution does not allow detailed discussion of the sugar conformation of AkAbfB.…”

“…The distances between these sulfur atoms and the C-4/C-5 atoms of arabinofuranose are about 4 Å, and this disulfide bond seems to recognize these carbon atoms through hydrophobic interaction. In GH51 GsAbfA, a tryptophan side chain replaces this disulfide bond and similarly recognizes the carbon atoms of arabinofuranose through hydrophobic interaction (15). A disulfide bond between adjacent cysteines joined by a cis-peptide bond has been rarely reported, i.e.…”

“…However, more detailed information for the binding specificity and affinity of the new CBM of GH54 is still needed to define its function and biological role. On the other hand, GH51 GsAbfA contains a non-catalytic ␤-sandwich domain, which exhibits structural similarity with non-catalytic domain C of ␣-amylases and also with cellulose-binding domains (15). However, no sugar-binding ability of the ␤-sandwich domain has been mentioned.…”

“…However, AwAbfI preferentially hydrolyzed the ␣-1,5-linkage of branched arabinotrisaccharide, whereas AwAbfII preferentially hydrolyzed an ␣-1,3-linkage in the same substrate and hydrolyzed the arabinosyl side-chain linkage of arabinan. The crystal structure and catalytic residues of GH51 Geobacillus stearothermophilus T-6 ␣-L-arabinofuranosidase A (GsAbfA) have been determined (15)(16)(17). On the other hand, the catalytic residues and three-dimensional structure of GH54 enzymes are completely unknown.…”

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

“…2a, c and e). Although the higher-order oligomeric form detected for both enzymes has an estimated molecular mass close to the pentamer, two lines of evidence suggest that this form corresponds to a hexamer: (i) the crystal structures of AbfA from G. stearothermophilus and Araf51 from C. thermocellum, which display 71 % and 63 % identity, respectively, to AbfA from B. subtilis, showed that they are organized as hexamers, trimers of dimers (Hovel et al, 2003;Taylor et al, 2006); (ii) the cross-linking experiments did not reveal a trimeric form, favouring the oligomerization as dimers and a higher-order structure with a mass greater than 250 kDa.…”