7EXV

GH127 beta-L-arabinofuranosidase HypBA1 covalently complexed with beta-L-arabinofuranoylamide

Summary for 7EXV

• Entry DOI: 10.2210/pdb7exv/pdb
• Related: 7EXU
• Descriptor: Non-reducing end beta-L-arabinofuranosidase, ZINC ION, 2-bromanyl-N-[(2S,3R,4R,5S)-5-(hydroxymethyl)-3,4-bis(oxidanyl)oxolan-2-yl]ethanamide, ... (4 entities in total)
• Functional Keywords: (alpha/alpha)6 barrel, glycoside hydrolase family 127, hydrolase
• Biological source: Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
• Total number of polymer chains: 1
• Total formula weight: 74793.39

Authors

Sawano, K.,Arakawa, T.,Yamada, C.,Fujita, K.,Fushinobu, S. (deposition date: 2021-05-28, release date: 2021-11-17, Last modification date: 2023-11-29)

Primary citation

Maruyama, S.,Sawano, K.,Amaki, S.,Suzuki, T.,Narita, S.,Kimura, K.,Arakawa, T.,Yamada, C.,Ito, Y.,Dohmae, N.,Fujita, K.,Ishiwata, A.,Fushinobu, S.
Substrate complex structure, active site labeling and catalytic role of the zinc ion in cysteine glycosidase.
Glycobiology, 32:171-180, 2022

PubMed Abstract: β-l-Arabinofuranosidase HypBA1 from Bifidobacterium longum belongs to the glycoside hydrolase family 127. At the active site of HypBA1, a cysteine residue (Cys417) coordinates with a Zn2+ atom and functions as the catalytic nucleophile for the anomer-retaining hydrolytic reaction. In this study, the role of Zn2+ ion and cysteine in catalysis as well as the substrate-bound structure were studied based on biochemical and crystallographic approaches. The enzymatic activity of HypBA1 decreased after dialysis in the presence of EDTA and guanidine hydrochloride and was then recovered by the addition of Zn2+. The Michaelis complex structure was determined using a crystal of a mutant at the acid/base catalyst residue (E322Q) soaked in a solution containing the substrate p-nitrophenyl-β-l-arabinofuranoside. To investigate the covalent thioglycosyl enzyme intermediate structure, synthetic inhibitors of l-arabinofuranosyl haloacetamide derivatives with different anomer configurations were used to target the nucleophilic cysteine. In the crystal structure of HypBA1, β-configured l-arabinofuranosylamide formed a covalent link with Cys417, whereas α-configured l-arabinofuranosylamide was linked to a noncatalytic residue Cys415. Mass spectrometric analysis indicated that Cys415 was also reactive with the probe molecule. With the β-configured inhibitor, the arabinofuranoside moiety was correctly positioned at the subsite and the active site integrity was retained to successfully mimic the covalent intermediate state.

PubMed: 34735571
DOI: 10.1093/glycob/cwab103

Experimental method

X-RAY DIFFRACTION (2.6 Å)