Crystal structure of an Exo-1,5-{alpha}-L-arabinofuranosidase from Streptomyces avermitilis provides insights into the mechanism of substrate discrimination between exo- and endo-type enzymes in glycoside hydrolase family 43.
Exo-1,5-α-L-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-L-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-L-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-L-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite -1, formed by the flexible loop region Tyr-281-Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.
Fujimoto
Zui
Z
Protein Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Ichinose
Hitomi
H
Maehara
Tomoko
T
Honda
Mariko
M
Kitaoka
Motomitsu
M
Kaneko
Satoshi
S
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods*; D006026:Glycoside Hydrolases / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D001120:Arginine; D006026:Glycoside Hydrolases