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

Hirofumi Shoun,Hiroshi Matsuzawa,Akimasa Miyanaga,Shinya Fushinobu,Takayoshi Wakagi,Takuya Koseki

doi:10.1074/jbc.m405390200

Abstract

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 alpha-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a beta-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a beta-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu(221) and Asp(297), respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.

Highlights

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 ␣-L-arabinofuranosidase (AkAbfB)
According to the classification of carbohydrate-binding module (CBM) defined by Boraston et al [49], arabinose-binding domain (ABD) can be grouped into the fold family 2 (␤-trefoil) along with CBM13
The details of the substrate recognition type of ABD is still unknown, it appears to be grouped into the type C small sugar binding CBMs because at least three hydrogen bonds are formed in the small arabinose binding pocket of each subdomain

Summary

EXPERIMENTAL PROCEDURES

Data Collection—Expression, purification, crystallization, and x-ray data collection of native and xenon-derivatized crystals were previously reported [18]. A platinum derivative was prepared by soaking native crystals in 15 mM K2PtCl4 for 2 days. A mercury derivative was prepared by soaking in 10 mM HgCl2 for 2 days. The arabinose complex was prepared by soaking native crystals in 50 mM L-arabinose for 90 min. The data sets were collected at 100K with a CCD camera on the NW12 stations at Photon Factory AR, High Energy Accelerator Research Organization, or with an R-axis IVϩϩ system on a x-ray generator, UltraX18 (Rigaku Corp.). The arabinose complex structure was solved by starting from the refined native structure. The mutations were confirmed by determining the nucleotide sequences. These plasmids were transformed into Pichia pastoris GS115, and the mutant enzymes were expressed, purified, and assayed as described previously [18]

RESULTS

Basic fibroblast growth factor mutant

DISCUSSION