Since glycoside hydrolase (GH) family 127 was created according to the discovery of β-L-Araf-ase HypBA1 from Bifidobacterium longum in 2011, more than 2,500 ORFs are currently classified into GH127 and its related family, GH146. However, only a few enzymes in these families have been characterized. We have previously reported the crystal structure of HypBA1 complexed with β-L-Araf, representing the product complex state. The active site has a unique Zn2+ center coordinated by Cys×3 + Glu. Among the active site residues, Cys417 was suggested to be the nucleophile residue. Further studies revealed that the Cys×3 + Glu active site structure is completely conserved among the characterized GH127-GH146 enzymes so far. To examine the hypothetical reaction mechanism of HypBA1, a synthetic substrate (pNP-β-L-Araf) and site-specific probes for Cys (haloacetamide derivatives of L-Araf) were soaked into HypBA1 crystals. We obtained a crystal structure of an acid/base catalyst residue mutant (E388Q) complexed with pNP-β-L-Araf, representing the Michaelis complex state. A complex structure with BrAcNH-β-L-Araf revealed that the amide-β-L-Araf moiety was covalently attached to Cys417.

Structural analysis for studying the reaction mechanism of β-L-arabinofuranosidases

2019 Fall National Meeting

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