Structural basis for the strict substrate specificity of β-D-galactofuranosidase fromStreptomycessp. JHA19

Abstract D-Galactofuranose (Galf) is widely distributed in polysaccharides and glycoconjugates of bacteria, filamentous fungi, and protozoa. The biosynthetic and degradation pathways of Galf in pathogens have attracted attention as potential targets for drug development. β-D-Galactofuranosidase (Galf-ase) releases Galf from the non-reducing ends of glycans. Galf-ase activity is often exhibited by α-L-arabinofuranosidases, which hydrolyze a similar substrate. Several Galf-specific Galf-ases that cleave only Galf and not L-arabinofuranose (Araf) have recently been identified in the glycoside hydrolase (GH) families 2, 5, and 43. However, the structural basis of how they discriminate the substrates is unknown. ORF1110, belonging to GH2, is the first identified Galf-specific Galf-ase isolated from Streptomyces sp. JHA19. Here, we solved the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, D-iminogalactitol (Ki = 65 μM). ORF1110 binds to the C5-C6 hydroxy groups of D-iminogalactitol with an extensive and integral hydrogen bond network. This result suggests that in the case of Araf, which lacks the C6 hydroxymethyl group, this network is not formed. The domain structure of ORF1110 is similar to that of β-glucuronidases and β-galactosidases, which belong to the same GH2 family and hydrolyze pyranose substrates. However, their active site structures were completely different. A predicted structure of the C-terminal Abf domain of ORF1110 was very similar to the carbohydrate-binding module family 42, which binds Araf, and pockets that may bind Galf were present. Competing Interest Statement The authors have declared no competing interest. Footnotes Conflict of interest The authors declare no competing financial interests. Abbreviations - Galf - D-Galactofuranose - Galf-ase - β-D-Galactofuranosidase - GH - glycoside hydrolase - CAZy - Carbohydrate-Active enZyme - Araf-ase - α-L-arabinofuranosidase - pNP - p-nitrophenyl - IGT - D-iminogalactitol - SeMet - selenomethionine - RMSD - root mean square deviation - CBM - Carbohydrate-Binding Module