A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases

Abstract

The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but noncarbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 and 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regard to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multigene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii . We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate-binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs and cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment.

Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum

“…Whether this is the case, in a similar vein to gut species enabling cross-feeding on complex carbohydrates or not remains to be elucidated (40), but it is however likely that the C. butyricum tannases facilitate its competitiveness in tannin rich environments. Previous studies on microorganisms encoding multiple esterase enzymes of the same type, such as feruloyl and glucuronyl esterases, have shown that the enzymes typically display quite different substrate preferences (41,42), and similarly this first study of multiple tannases from the same microorganism follows that trend. Future studies on the multiplicity J o u r n a l P r e -p r o o f of tannases in different microorganisms will help further shed light on specific biological roles and possibly also lead to improved utilization of these enzymes in industrial settings.…”

Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

“…Deducing the preferred substrate of a multicatalytic enzyme can be challenging due to the highly specialized nature of these proteins and the vast diversity among polysaccharides, especially in the context of the complex cell wall polymer network. A lack of intramolecular enzyme synergy has also been observed for other multicatalytic enzymes, such as Fj CE6-CE1 from F. johnsoniae 20 , Ck Xyn10C-GE15A from C. kristjansonii 32 , and Dm CE1B from Dysgonomonas mossii 31 . Given the complexity of the substrates targeted by these enzymes, which are presumed to be part of LCCs, it is currently unclear whether the lack of observed intramolecular enzyme synergy is the result of missing intramolecular synergy, a lack of the right substrate, or another unknown reason.…”

“…The reason for combining activities presumed to target complex LCCs and shorter XOs into one single enzyme is however not clear. To gain further insight into the function of Be CE15A-Rex8A, as well as its truncated single domain versions ( Be CE15A and Be Rex8A), the enzymes were assayed for their ability to boost the action of a commercially available GH11 xylanase (Xyn11A), which has previously been used successfully in similar experiments 20 , 31 . Ball-milled corn cob biomass, which has a high content of GAX 15 , was used as substrate.…”

“…For example, the CAZymes CelA (N-terminal glycoside hydrolase family 9 (GH9) endo -cellulase and C-terminal GH48 exo -cellulase) from Caldicellulosiruptor bescii 29 , ChiA (N-terminal exo - and C-terminal endo -chitinase domain) from Flavobacterium johnsoniae 16 , 30 , and Bo CE6-CE1 (acetyl-feruloyl esterase) from Bacteroides ovatus 20 , have significantly enhanced substrate turnover capabilities as full-length enzymes compared to their individual catalytic domains. In some cases, intramolecular synergy has however not been observed for multicatalytic enzymes 20 , 31 , 32 . This might be caused by a lack of appropriate substrates, analytics, or appropriate reaction conditions, or simply that there is no synergy between the catalytic domains.…”

Tracking the gastrointestinal digestive and metabolic behaviour of Dendrobium officinale polysaccharides by fluorescent labelling

“…11,12 The metabolism of polysaccharides in Bacteroides is regulated by polysaccharide utilization loci (PULs). 13,14 Various genera of Bacteroides are able to compile specific glycoside hydrolases (GH), polysaccharide lyases (PL) and glycosyltransferases (GT) for the metabolism of natural polysaccharides by recognizing polysaccharide-specific glycosidic bond linkages and upregulating the expression of the corresponding PUL genes. 15,16 Luis, A. S. et al found that α-mannanase and arabinoxylan were expressed by the intestinal B. thetaiotaomicron and Bacteroides.…”