A novel glycoside hydrolase family 97 enzyme: Bifunctional β-l-arabinopyranosidase/α-galactosidase from Bacteroides thetaiotaomicron

Abstract

Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, α-glucoside hydrolase and α-galactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 α-glucoside hydrolase and α-galactosidase, were characterized in this study. BT3664 was identified to be an α-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both β-l-arabinopyranoside and α-d-galactopyranoside, and thus we designate BT3661 as a β-l-arabinopyranosidase/α-d-galactosidase. Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 α-galactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears Glu361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The k/K value for the hydrolysis of p-nitrophenyl α-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the k/K value for hydrolysis of p-nitrophenyl β-l-arabinopyranoside.

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

“… 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. The estimated molecular masses of these detected proteins were about 120 kDa, which is comparable to that determined by SDS-PAGE.…”

α-Galactosidase and Sucrose-Kinase Relationships in a Bi-functional AgaSK Enzyme Produced by the Human Gut Symbiont Ruminococcus gnavus E1

“…In the CAZy database 1 , the bacterial enzymes responsible for the degradation of RFOs, α-galactosidases, are classified into the glycoside hydrolase (GH) families 4, 27, 36, 57, 97, and 110, but mainly gathered within the families GH27 and GH36 (Lombard et al, 2014). Over the last 15 years, the structures of several α-galactosidases have been determined within the GH27, GH36, and GH97 families (Fujimoto et al, 2003(Fujimoto et al, , 2009Garman and Garboczi, 2004;Golubev et al, 2004;JCSG., 2005JCSG., , 2013Comfort et al, 2007;Lieberman, 2009;Okuyama et al, 2009;Fernández-Leiro et al, 2010;Guce et al, 2010;Vorobiev et al, 2010;Bruel et al, 2011;Fredslund et al, 2011;Merceron et al, 2012;Yu et al, 2014;Adamson et al, 2016;Kikuchi et al, 2017;Kytidou et al, 2018;Hobbs et al, 2019;Rowland et al, 2019). The α-galactosidases from GH27 and GH36 families share a common catalytic domain characterized by a (β/α) 8 -barrel folding type and a C-terminal domain composed of β-sheets.…”

Characterization of a GH36 β-L-Arabinopyranosidase in <i>Bifidobacterium adolescentis</i>

“… 11) GH97 β-L-arabinopyranosidase has recently been cloned from Bacteroides thetaiotaomicron . 12) Because of the structure similarity between β-L-Ara p and α- D- galactopyranose (α-Gal), α- D- galactopyranosidase (EC 3.2.1.22) can potentially possess a β-L-arabinopyranosidase activity. In fact, almost all β-L-arabinopyranosidases belonging to GH27 and GH97 have α- D- galactopyranosidase activity.…”