Microbial pectin recognition and utilization of the mammalian gastrointestinal tract

Abstract

Plant cell wall polysaccharides represent one of the major nutrients available to the human microbiota, which has a significant impact on host nutrition and health. Pectins are one of the major plant cell wall components. Understanding the mechanism of protein recognition and enzymatic degradation of these polysaccharides can have significant implications, not only in promotion of human health, but also in an industrial context. In this thesis the founding member of a carbohydrate binding module (CBM) family that targets homogalacturonan (HG) was characterized. The mechanism of degradation of rhamnogalacturonan-I and II (RG-I and RG-II) by Bacteroides thetaiotaomicron, a member of human microbiota, was also explored. Efficient plant cell wall degradation requires a close association between catalytic modules with CBMs. In Chapter 3, the characterization of Ruminococcus flavefaciens CBM77PL1/9 revealed that this CBM specifically binds to non-methylated HG. The structural characterization disclosed a new binding mechanism where the positively charged residues (lysines) interact with the negative carboxyl groups present in the ligand. Additionally, a dimer of this CBM has shown to be a suitable probe to target pectins in a plant cell wall context. B. thetaiotaomicron is able to grow on pectins. In this study, a model for RG-I and RG-II degradation was described. The cleavage of RG-II is achieved by a hierchical exo mode of degradation. Chapter 4 showed that the degradation of Chain B requires eight enzymes where the -L-rhamnosidases, BT0986 and BT1019, specifically target the linkages 1,2 and 1,3-arabinopyranose, respectively. It was shown that the linkage between L-rhamnose and D-apiose is an -linkage, likely 1,3’. BT1001, which targets the rhamnose-apiose glycosidic bond requires the cleavage of side chain and backbone, suggesting that this is the last linkage cleaved in RG-II degradation. The structural and biochemical characterization of a GH106 enzyme (BT0986) revealed a calcium dependent enzyme with a catalytic apparatus consistent with an inverting mechanism. The structure of a GH127 enzyme (BT1003) involved in RG-II showed that the aceric acidase lacks the residue that is the proposed catalytic acid/base in GH127 -L-arabinofuranosidases. This may indicate that the aceric acidase cleaves glycosidic bonds by a mechanism that differs from other enzymes in the GH127 family. In Chapter 5, key enzymes in the RG-I degradation system were characterized. Three polysaccharide lyases (PLs) (BT4170, BT4175 and BT4183) revealed complementary specificities. BT4170, identified on the cell surface, was shown to be essential for RG-I backbone utilization. Four GH28 enzymes were characterized: BT4149 and BT4153 target the RG-I backbone displaying distinct substrate specificities based on the degree of polymerization of the oligosaccharide. BT4155 removes the HG remnants attached to RG-I and BT4146 is specific for a RG-I backbone disaccharide. The surface glycan binding protein BT4167 has shown to recognize RG-I. The structural characterization of BT4170 (PL9) revealed that the residues implicated in substrate recognition in this rhamnogalacturonan lyase are not conserved in PL9 pectate lyases. The proposed model of how 13 enzymes encoded by B. thetaiotaomicron mediate the degradation of RG-I contributes to our knowledge of pectin degradation.