Abstract
Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.
Highlights
The human large intestine is home to a large microbial community termed the human gut microbiota (HGM), which has substantial impact on the health and physiology of its host
Bacteroides thetaiotaomicron is a prominent member of the HGM, equipped with a large repertoire of carbohydrate-active enzymes (CAZymes) and considered as a generalist being able to forage on a wide range of dietary or host glycans
Both sugars were purified by size-exclusion chromatography and treated independently with a mixture of recombinant mixture of five enzymes including BT1017 (A5) enzymes including BT1017, BT1018 (a-galacturonidase), BT1021 (a-arabinofuranosidase), BT1012 (b-apiosidase), and BT1001 (a-rhamnosidase), which target specific glycosidic linkages in RG-II [5]
Summary
The human large intestine is home to a large microbial community termed the human gut microbiota (HGM), which has substantial impact on the health and physiology of its host. Among the B. thetaiotaomicron repertoire, several founding members of novel CAZyme families were characterized including a pectin methylesterase (PME) BT1017. When cultured in media containing extensively purified apple RG-II as a sole carbon source, a B. thetaiotaomicron genetic mutant lacking the BT1017 enzyme (DBT1017) produces a pentasaccharide Rhaa1,3-Api-b1,2-(Araf-a1,3)-(6-O-Me-GalA-a1,4)-GalA here referred to as DBT1017oligoA (Fig. 1A) [5]. A methylesterase essential for pectin metabolism (b-apiosidase) cleaves the linkage between Api and the reducing end GalA; and BT1001 (a-rhamnosidase) cleaves the linkage between Rha and Api in the Rha–Api disaccharide (Fig. 1B). We showed that the B. thetaiotaomicron mutant DBT1017, when cultured in carbohydrate extract from apple juice (CEAJ) as a sole carbon source, generates a second oligosaccharide (hereafter referred to as DBT1017oligoB). We revealed that BT1017 is a serine esterase with an a/b-hydrolase fold and has not evolved from the progenitor protein that gave rise to the CE8 family of PMEs, which are predominantly comprised of right-handed b-helices
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