Innovating glycoside hydrolase activity on a same structural scaffold

Abstract

Carbohydrates play many fundamental roles in the cell physiology and development of plants, animals, and microbes. They can take the form of glycoproteins, glycolipids, and polysaccharides and represent the largest reservoir of carbon resources that are fueling microbial communities as well as free-living microorganisms. The structural diversity of naturally occurring carbohydrate compounds is matched by an equally diverse class of enzymes tailored specifically to break down each and every glycosidic bond. The enzymes responsible for the hydrolysis of these glycosides are termed glycoside hydrolases (GHs), or glycosidases, and are grouped and classified in sequence-based families within the "Carbohydrate-Active enZYmes" Database (1). In general, they are very efficient catalysts that can enhance the reaction rate by an order of 10 17 -fold over the noncatalytic reaction rate (2). The mechanism by which they achieve this feat has thus been the subject of extensive work (reviewed in refs. 3, 4). The outcome of the hydrolytic reaction, in most cases involving a couple of carboxylic acid residues positioned to activate a water molecule that is added to the glycosidic bond, occurs either with net retention or inversion of the stereochemistry at the anomeric center, and the canonical mechanisms explaining these actions were described as early as 1953 in a seminal paper by Koshland (5). However, in recent years, the flow of new (bacterial) sequences and families has provided several new and unusual mechanisms (6), highlighting the variability of these catabolic processes but also the knowledge gap that still exists in the field of glycobiochemistry. In particular, the recent advent of systems encoded by polysaccharide utilization loci (PULs) in Bacteroidetes has revealed an elegant way of discovering new families and functions (7). 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, 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. Even more intriguingly, the catalytic active site is not, as usual, located in the center of the deep pocket formed on the anterior side of the beta-propeller fold but is found instead on the "backside" of the enzyme.Seeking for enzymes in Bacteroides thetaiotaomicron that are able to degrade rhamnose-containing complex carbohydrates, such as arabino-galactan proteins (AGPs) or rhamnogalacturonans I and II (RGI and RGII), the authors inspected the up-regulation of specific PULs in the presence of these substrates and screened those proteins for activities that had no annotated functions. In so doing, they discovered that BT...

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“…This could be because the former is a propionate-producing bacteria, as also reported by Wang et al ( 47 ). Bacteroides thetaiotaomicron , an obligate anaerobe colonizing the gastrointestinal food particles and mucus, forms several polysaccharide utilization loci (PULs) to break down numerous dietary polysaccharides and provide genes to promote the acquisition and utilization of carbohydrates ( 45 , 50 ). Evidence has shown that Bacteroides thetaiotaomicron could activate different genes to produce proteins attaching the polysaccharide-rich food surface and to degrade polysaccharides ( 51 ).…”

Sulfation of Arabinogalactan Proteins Confers Privileged Nutrient Status to Bacteroides plebeius

“…The crystal structure of the enzyme in complex with the reaction product, glucuronic acid, together with biochemical analysis of appropriate mutants, showed that the posterior surface houses an active site of the seven-bladed β-propeller enzyme ( 30 ). This was surprising, as the catalytic center of all other β-propeller enzymes, including several CAZymes, is located on the anterior surface ( 31 ). In addition, the central catalytic residue in the posterior active site, His48 in BtGH145, is not invariant in GH145 ( Table S2 in the supplemental material), and those enzymes lacking the histidine were shown not to display rhamnosidase activity against GA-AGP.…”

“…Bacteroides spp. were routinely cultured under anaerobic conditions at 37°C using an anaerobic cabinet (Whitley A35 workstation; Don Whitley) in culture volumes of 0.2, 2, or 5 ml of TYG (tryptone-yeast extract-glucose medium) or minimal medium (MM) ( 31 ) containing 0.5 to 1% of an appropriate carbon source and 1.2 mg ml −1 porcine hematin (Sigma-Aldrich) as previously described ( 10 ). The growth of the cultures was monitored by optical density at 600 nm (OD 600 ) using a Gen5 v2.0 microplate reader (Biotek).…”