Ascertaining the biochemical function of an essential pectin methylesterase in the gut microbe Bacteroides thetaiotaomicron

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotao- micron 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 D BT1017oligoB generated by a BT1017 deletion mutant ( D BT1017) during growth on carbohydrate extract from apple juice. Structural analyses of D BT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR b a b 1,2-(Ara - a a 1,4)-GalA) RG-II -hydrolase

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 DBT1017oligoB generated by a BT1017 deletion mutant (DBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of DBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-b1,4-(2-O-Me-Xyl-a1,3)-Fuc-a1,4-(GalA-b1,3)-Rha-a1,3-Api-b1,2-(Araf-a1,3)-(GalA-a1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an a/b-hydrolase fold, consisting of a central twisted 10-stranded b-sheet sandwiched by several a-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed b-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.
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. Pectins, which are a major component of plant-based diets, have been shown to exert a significant selective pressure on HGM species (1-3) and hence have great potential as tools to manipulate the HGM. Pectins are defined as D-galacturonic acid-containing plant cell wall polysaccharides. The pectin macrostructure consists of three major polysaccharides: rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-II), and homogalacturonan (4). Of these, RG-II is the most complex, consisting of several heterogenous side chains (A, B, C, D, E, and F), which are linked to a backbone of D-galacturonic acid (GalA) residues ( Fig. 1A) (5). In total, RG-II contains at least 22 distinct glycosidic linkages and 13 different monosaccharides (Fig. 1A). The structure of RG-II is highly conserved; however, there is some variation in RG-II between plant species particularly at the termini of side chain B and in the methylation pattern of side chain A, as described previously (6, 7).
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 (for a review see Ref. 4). B. thetaiotaomicron has the ability to cleave 21 of the 22 glycosidic linkages in RG-II (except that in the disaccharide 2-O-Me-Xyl-a1,3-Fuc) (5). Among the B. thetaiotaomicron repertoire, several founding members of novel CAZyme families were characterized including a pectin methylesterase (PME) BT1017. BT1017 was shown to remove the 6-O-methyl decoration of GalA in the homogalacturonan backbone of RG-II, therefore playing a critical role in enabling access to the rest of the RG-II structure by other RG-II-degrading enzymes (5). Currently more than 18 carbohydrate esterase families have been assigned, according to the CAZyme database (8); this topic was recently reviewed by Nakamura et al. (9). Of the 18 families, CE8 is the only family that contains PMEs. BT1017, however, displays no sequence similarity to CE8 esterases, and hence the structural basis for its catalytic function is unknown. 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 Rha-a1,3-Api-b1,2-(Araf-a1,3)-(6-O-Me-GalA-a1,4)-GalA here referred to as DBT1017oligoA (Fig. 1A) (5). The complete degradation of DBT1017oligoA requires five enzymes (BT1017, BT1018, BT1021, BT1012, and BT1001) collectively referred to here as A5 (Fig. 1B). BT1017 cleaves the 6-O-methylester linkage from the backbone GalA; BT1018 (a-galacturonidase) cleaves the glycosidic linkage between the two backbone GalA residues; BT1021 (a-arabinofuranosidase) cleaves the linkage between Araf (chain F) and the reducing end GalA; BT1012 (b-apiosidase) cleaves the linkage between Api and the reducing end GalA; and finally BT1001 (a-rhamnosidase) cleaves the linkage between Rha and Api in the Rha-Api disaccharide (Fig. 1B).
In the present study, 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). Our structural analyses revealed that DBT1017oligoB is a dimethylated nonasaccharide containing components of the RG-II backbone and its side chains. We characterized the kinetic properties of BT1017, showing that the enzyme has a low turnover against apple RG-II, DBT1017oligoA, and DBT1017oligoB and hence may represent a rate-limiting step during RG-II metabolism. We revealed that BT1017 is a serine esterase with an a/b-hydrolase fold and hence 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.

Characterization of DBT1017oligoB
B. thetaiotaomicron DBT1017 deletion mutant was cultured on CEAJ for 48 h to stationary phase (A 600 nm ;1.0), and TLC was first used to analyze the culture supernatants. The data showed that DBT1017 generates two oligosaccharides, defined as DBT1017oligoA and DBT1017oligoB ( Fig. 2A). Both sugars were purified by size-exclusion chromatography and treated independently with a mixture of recombinant 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). Unless otherwise stated, all the recombinant RG-IIdegrading enzymes mentioned in this text were the same constructs used by Ndeh et al. (5) and lack the N-terminal signal peptide (SP) regions (Fig. 1C). The products of the enzymatic treatment were then analyzed by TLC and HPLC (Fig. 2, B and C). Digestion of DBT1017oligoA yielded GalA, Araf, Rha and Api indicating that the molecule is the methylated pentasaccharide (Rha-a1,3-Api-b1,2-(Ara-a1,3)(6-O-Me-GalA-a1,4)-GalA, which was used to demonstrate the site of action of the PME in Ndeh et al. (5). The digestion of DBT1017oligoB, on the other hand, was incomplete, yielding only two monosaccharides (Araf and GalA) and a third product of unknown identity (Fig. 2, B and C). The release of GalA and Araf from DBT1017oligoB by DBT1017oligoA-specific enzymes BT1018 (a-galacturonidase) and BT1021 (a-arabinofuranosidase) suggests that DBT1017oligoB contains the backbone GalA and the side-chain F Araf sugars characteristic of DBT1017oligoA (Fig.  1, A and B). To determine the full structure of DBT1017oligoB, a combination of MS, enzymatic and NMR analyses were performed. First, MS data revealed that DBT1017oligoB has a protonated molecular mass ([M 1 H] 1 ) of 1453.44 Da (Fig. 3A). When treated with BT1017, the mass of DBT1017oligoB decreased by 28.03 Da (Fig. 3B), corresponding to the loss of two methyl groups. This suggests that DBT1017oligoB contains two esterlinked methyl groups that were hydrolyzed by the BT1017 PME. Second, when WT B. thetaiotaomicron was cultured on DBT1017oligoB, the bacterium accumulated the disaccharide 2-O-Me-Xyl-a1,3-Fuc, which is unique to side chain A of RG-II but not present in DBT1017oligoA (Fig. 3C). The sugar Api was also detected. These results demonstrate that DBT1017oligoB contains components of DBT1017oligoA and additional sugars from RG-II side chain A. Last, DBT1017oligoB was shown to be susceptible to attack by the b-D-glucuronidase enzyme BT0996, which released GlcA (Fig. 3D). Because this required pretreatment with BT1017, this result suggests that at least one of the methyl decorations in DBT1017oligoB sterically hinders the activity of BT0996. Release of free GlcA is also an indication that DBT1017oligoB lacks the terminal L-Gal residue, which is a1,2linked to GlcA at the nonreducing end of chain A (Fig. 1A).
Both sugars differ by the presence of either a1,2or b1,3linked GalA (underlined). To determine which of them corresponded to DBT1017oligoB-2Me, enzymes targeting all linkages in the predicted sugars (DBT1017oligoB-2Me-a and DBT1017oligoB-2Me-b) were used to sequentially digest DBT1017oligoB. The first set of recombinant enzymes collectively referred to here as B5 enzymes include BT1017, BT1018, BT1021, BT0996, and BT1012. These together should cleave the two ester groups, the backbone a1,3-linked GalA, the side chain F a1,3-linked Araf, the side chain A b1,4-linked GlcA, and the reducing end/backbone GalA residue, respectively (  absence of either a1,2or b1,3-linked GalA (underlined). Digestion of DBT1017oligoB-2Me with a mixture of recombinant B5 enzymes (BT1017, BT1018, BT1021, BT0996, and BT1012) and subsequent analyses by TLC revealed the generation of a product that migrates to a similar extent as the sugar standard MXFGRA-b (Fig. 4A, lane 8 in white rectangle). However, it was also possible that the product corresponded to MXFGRA-a because of the significant structural similarity to MXFGRA-b. As a result, it was referred to as MXFGRA-x. Both a1,2and b1,3-GalA linkages in MXFGRA-a and MXFGRA-b have been shown to be specifically targeted by the enzymes BT0997 (a-galacturonidase) and BT0992 (b-galacturonidase), respectively (5) (Fig. S1, D and E); hence to determine whether the product contained aor b-linked GalA residues, each of these enzymes (BT0992 and BT0997) was used to further digest MXFGRA-x. TLC analyses of the reaction showed that MXFGRA-x was digested by BT0992 but not by BT0997 (Fig. 4A, lanes 9 and 10, respectively), indicating that the exposed GalA residue in the product was b1,3-linked to Rha and that the pentasaccharide was MXFGRA-b. This was also confirmed by 2D HSQC NMR analyses of DBT1017oligoB, which detected 1 H and 13 C HSQC anomeric signals (d H 4.67 and d C 104.1) of b-GalA. The NMR analyses also revealed H1/ C1 signals of all other carbohydrate residues in the anomeric region of the spectrum. These include signals for GalA-a1-4, Araf-a1-3, Api-b1-2, Rha-a1-3, Fuc-a1-4, 2-O-Me-Xyl-a1-3, and GlcA-b1,4 (Fig. 4B), which were assigned by comparison with published data (10). Two weaker cross-peaks could be assigned to the anomeric center of the reducing-end GalA residue in the backbone of DBT1017oligoB. The full monosaccharide composition of DBT1017oligoB-2Me was confirmed by treatment of the sugar with a combination of A5 enzymes together with BT0996, BT0992, and other RG-II-degrading enzymes BT1002 (a-L-fucosidase) and BT1001 (a-L-rhamnosidase) and analyses of the digested sample by HPLC. The results showed that the enzymes degraded the sugar into all its constituent monosaccharides GlcA, GalA Rha Api Araf, and the disaccharide 2-O-Me-Xyl-a1,3-Fuc (Fig. 4C). A model showing the cleavage sites of various enzymes on DBT1017oligoB is shown in Fig. 4D.