Unusual active site location and catalytic apparatus in a glycoside hydrolase family

Abstract

The human gut microbiota use complex carbohydrates as major nutrients. The requirement for an efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that these bacteria represent a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis, we focused on enzymes that hydrolyze rhamnosidic bonds, as cleavage of these linkages is chemically challenging and there is a paucity of information on L-rhamnosidases. Here we screened the activity of enzymes derived from the human gut microbiota bacterium Bacteroides thetaiotaomicron, which are up-regulated in response to rhamnose-containing glycans. We identified an α-L-rhamnosidase, BT3686, which is the founding member of a glycoside hydrolase (GH) family, GH145. In contrast to other rhamnosidases, BT3686 cleaved L-Rha-α1,4-D-GlcA linkages through a retaining double-displacement mechanism. The crystal structure of BT3686 showed that the enzyme displayed a type A sevenbladed β-propeller fold. Mutagenesis and crystallographic studies, including the structure of BT3686 in complex with the reaction product GlcA, revealed a location for the active site among β-propeller enzymes cited on the posterior surface of the rhamnosidase. In contrast to the vast majority of GH, the catalytic apparatus of BT3686 does not comprise a pair of carboxylic acid residues but, uniquely, a single histidine functions as the only discernable catalytic amino acid. Intriguingly, the histidine, His48, is not invariant in GH145; however, when engineered into structural homologs lacking the imidazole residue, α-L-rhamnosidase activity was established. The potential contribution of His48 to the catalytic activity of BT3686 is discussed.rhamnosidase | human gut microbiota | gum arabic | Bacteroides thetaiotaomicron | catalytic histidine

Innovating glycoside hydrolase activity on a same structural scaffold

“…Instead of being located in the usually conserved active site pocket on the anterior side of the beta-propeller, however, the monosaccharide was bound in a shallow cleft on the posterior side of the enzyme. By site-directed mutagenesis, Munoz-Munoz et al (8) confirmed that this posterior cleft was indeed the active site of this new family of GHs. This result is outstanding, because in the majority of cases, the positions of active sites are conserved within a given fold (9,10).…”

“…A catalytic histidine remains a very uncommon catalytic residue for GHs and has previously been suggested to play a role in members of family GH117 α-1,3-(3,6-anhydro)-L-galactosidases (13,14), where it replaces the catalytic acid base that protonates the oxygen of the glycosidic linkage. In the mechanism proposed by Munoz-Munoz et al (8), however, the histidine residue is the only catalytic residue and abstracts the proton at the O2 position, implying that an epoxy intermediate is formed. This reaction intermediate, to date, is rare in carbohydrates and has been proposed to occur also in family GH99 α-mannosidases (15).…”

“…This reaction intermediate, to date, is rare in carbohydrates and has been proposed to occur also in family GH99 α-mannosidases (15). The mechanism of these enzymes is dependent on the stereochemical configuration of the substrate molecules that have in common the axial position of the hydroxyl group next to the anomeric carbon, opposite to the glycosidic bond that is cleaved in the catalytic reaction (8,15). Other unique and rare catalytic reaction mechanisms of GHs that depend on the nature of the substrate have also been observed in sialidases, where a conserved tyrosine plays a crucial role in the nucleophilic attack of the anomeric carbon (16).…”

“…It appears that variations of the classical reaction mechanisms depend on the nature of the substrate and are generally observed for particularly recalcitrant substrates, substrate-assisted reactions, or stereochemically challenging reactions, such as those reactions involving mannose or rhamnose (because the position of the hydroxyl group next to the anomeric carbon is axial). In PNAS, Munoz-Munoz et al (8) add an astonishing exception to the list of noncanonical GHs. They describe the discovery of a new GH family that, surprisingly, in addition possesses a very uncommon catalytic apparatus.…”

“…In so doing, they discovered that BT3686 released α-L-rhamnose units from the AGP substrate called gum arabic. As part of the thorough biochemical characterization, Munoz-Munoz et al (8) determined the 3D crystal structure of the newly discovered enzyme followed by site-directed mutagenesis, with the aim of elucidating the structural determinants responsible for the observed GH activity and α-L-rhamnose specificity. Unexpectedly, activity was not abolished when deleting the side chains of highly conserved residues within the anterior pocket formed by the sevenbladed beta-propeller fold, suggesting that this cleft was not the location of the active site.…”