The digestive machinery of a human gut bacterium: Structural enzymology of galactomannan utilisation

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Human gut bacteria utilise different types of polysaccharides present in our diet. One of these polysaccharides is galactomannan. Many organisms in the phylum Bacteroidetes have gene clusters encoding for all proteins required for hydrolysis, binding and transport of one type of polysaccharide, called polysaccharide utilisation loci (PULs). A PUL from the human gut bacterium Bacteroides ovatus was previously indicated to be involved in galactomannan utilisation (BoManPUL) and codes for one glycosde hydrolase (GH) family 36 α-galactosidase (BoGal36A, Paper I) and two GH26 β-mannanases (BoMan26A and BoMan26B, Papers II-IV).
In Paper I B. ovatus was shown to be able to grow on galactomannan, an ability which was lost upon knockout of BoManPUL, indicating that it was primarily responsible for galactomanna utilisation in this human gut bacteria. Papers I-III revealed a pathway for galactomanna utilisation in which BoMan26B initially cleaves polysaccharide substrates outside the cell, the products of which are transported into the periplasm and there further processed by BoGal36A, then BoMan26A.
BoMan26B is outer membrane attached, preferntially hydrolyses longer substrates (Paper II) and is one of the enzymes in GH26 least restricted by galactose substitutions (Papers II and III). Crystal structures revealed a long, open active site cleft, only being restricted by galactose substitutions in one subsite -2 and possibly favouring a substitution in subsite -4 (Paper III). BoMan26B provides sequential synergy to BoGal36A (Paper III), which preferentially hydrolyses internal galactose substitutions from galactomanno-oligosaccharides (Paper I). This hydrolysis of internal galactosyl units is unusual in GH36, which was shown to be caused by the absence of a loop that is present in other GH36 subgroup I members (Paper I). BoGal36A in turn provides sequential synergy to the endo-capable mannobiohydrolase BoMan26A, which preferentially cleaves unsubstituted mannooligosaccharides (Paper II). The structure of BoMan26A revealed a narrow active site cleft where at least two subsites are restricted by galactose substitutions, and which was blocked beyond subsite -2 by two loops: 2 and 8 (Paper II). NMR assignment of the BoMan26A backbone was carried out in Paper IV, to perform further NMR-based studies of substarte binding and protein dynamics relating to loops 2 and 8.
BoMan26A and BoMan26B differ both in structure and biochemistry and in a phylogenetic analysis of selected GH26 sequences they were shown to cluster in different branches (Paper III). The level of conservation around the active site cleft was generally low, with the exceptions of the -1 and +1 subsites (Paper III).
This thesis reveals a model for galactomannan PUL utilisation in B. ovatus, increasing the understanding of our gut microbiota. It also delves into the structure-function relationship of substrate specificity in, primarily, GH26 enzymes.

Details

Authors
  • Viktoria Bågenholm
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Other Chemistry Topics

Keywords

  • beta-mannanase, Glycoside hydrolase, enzymology, structural biology, kinetics, HPLC, product profiles, structure-function
Original languageEnglish
Awarding Institution
Supervisors/Assistant supervisor
Award date2019 Oct 19
Place of PublicationLund
Publisher
  • Lund University, Faculty of Science, Department of Chemistry
Print ISBNs978-91-7422-595-2
Electronic ISBNs978-91-7422-605-8
Publication statusPublished - 2018 Sep
Publication categoryResearch

Bibliographic note

Defence details Date: 2018-10-19 Time: 13:15 Place: Sal B, Kemicentrum, Naturvetarvägen 14, Lund External reviewer(s) Name: Leggio, Leila Lo Title: Professor Affiliation: Kemisk Institut, Köpenhamns universitet ---

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Research output: Contribution to journalArticle

Sumitha Krishnaswamyreddy, Viktoria Bågenholm, Nicholas A Pudlo, Hanene Bouraoui, Nicole M Koropatkin, Eric C Martens & Henrik Stålbrand, 2016 Jun 11, In: FEBS Letters. 590, 14, p. 2106-2118 13 p.

Research output: Contribution to journalArticle

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