Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases

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 472 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 616 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.

Identification and Characterization of Dextran α-1,2-Debranching Enzyme from <i>Microbacterium dextranolyticum</i>

“… 43) 44) 45) Kojibiose hydrolase, a member of GH65 that hydrolyzes α-(1→2)-glucosidic linkage in kojioligosaccharides, has recently been discovered in bacteria. 46) 47) A third protein was revealed to belong to GH97, where anomer-inverting α-glucoside hydrolases, anomer-retaining α-galactosidases, and anomer-retaining β-L-arabinopyranosidase have been found. 48) 49) 50) However, the GH97 protein lacks a putative signal peptide in contrast to the other two proteins found and shares a substantially larger degree of sequence identity with retaining α-galactosidases than with inverting α-glucoside hydrolases.…”

“…MdDDE shares 33 and 29 % total sequence identity with Flavobacterium johnsoniae GH65 kojibiose hydrolase (FjGH65A) 46) and human GH65 protein glucosylgalactosylhydrolxylysine α-glucosidase (HsPGGHG), 51) respectively. On the other hand, MdDDE exhibits a lower level of sequence identity (24 %) with Caldicellulosiruptor saccharolyticus GH65 kojibiose phosphorylase (CsKP, ABP66077.1).…”

“… 30) b Values were referred from Nakamura et al . 46) c α-Glucans produced by L. mesenteroides strains B-1298 and B-1397 contain 19 and 14 % α-(1→2) linkages at branch point, respectively. 30) d ND, not detected.…”

“…GH65 consists mainly of GPs from bacteria and GHs from eukaryotes that act on α-glucosidic linkages, 43) 44) 45) and recently the first bacterial GH, kojibiose hydrolase (EC 3.2.1.216), was discovered in Flavobacterium johnsoniae (FjGH65A) and Mucilaginibacter mallensis . 46) 47) Moreover, the biochemical and crystallographic studies of FjGH65A revealed catalytic residues and the anomer-inverting mechanism of GH65 GHs. 46) MdDDE was identified as a GH that released glucose from α-(1→2)-branched α-glucan in the present study and is therefore the second bacterial GH65 GH following kojibiose hydrolase.…”

“… 46) 47) Moreover, the biochemical and crystallographic studies of FjGH65A revealed catalytic residues and the anomer-inverting mechanism of GH65 GHs. 46) MdDDE was identified as a GH that released glucose from α-(1→2)-branched α-glucan in the present study and is therefore the second bacterial GH65 GH following kojibiose hydrolase. The sequence alignment with other GH65 enzymes revealed that both general acid (Glu666) and general base (Glu811) catalysts, which are essential for hydrolytic activity, are conserved ( Fig.…”