7FE3

Crystal structure of GH65 alpha-1,2-glucosidase from Flavobacterium johnsoniae

Summary for 7FE3

• Entry DOI: 10.2210/pdb7fe3/pdb
• Descriptor: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65, 1,2-ETHANEDIOL (3 entities in total)
• Functional Keywords: glycoside hydrolase, gh65, (alpha/alpha)6-barrel, kojibiose, dextran, hydrolase
• Biological source: Flavobacterium johnsoniae (strain ATCC 17061 / DSM 2064 / JCM 8514 / NBRC 14942 / NCIMB 11054 / UW101) (Cytophaga johnsonae)
• Total number of polymer chains: 3
• Total formula weight: 231081.43

Authors

Nakamura, S.,Miyazaki, T. (deposition date: 2021-07-19, release date: 2021-11-10, Last modification date: 2024-05-29)

Primary citation

Nakamura, S.,Nihira, T.,Kurata, R.,Nakai, H.,Funane, K.,Park, E.Y.,Miyazaki, T.
Structure of a bacterial alpha-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases.
J.Biol.Chem., 297:101366-101366, 2021

PubMed Abstract: Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.

PubMed: 34728215
DOI: 10.1016/j.jbc.2021.101366

Experimental method

X-RAY DIFFRACTION (1.54 Å)