Structural and biochemical characterisation of enzymes required for gram-negative capsule biosynthesis
Abstract
Bacteria are coated with a wide-variety of complex polysaccharides, including
lipopolysaccharide, crucial to their survival upon infection of a host organism. During
lipopolysaccharide biosynthesis, the integral membrane protein Wzy polymerises
undecaprenyl pyrophosphate-linked O-antigen repeat units. The mechanism of Wzycatalysed
polymerisation is unknown, as the difficulties associated with integral
membrane proteins have precluded its study. Here we describe a protocol that takes
membrane proteins from cloning to crystallisation trials, applied to 16 Wzy
homologues, 14 of which could be expressed and purified, with 5 entered into
crystallisation trials. The final O-antigen chain length is serotype-specific and is
controlled by Wzz via an unknown mechanism. Proposed mechanisms are based on a
direct interaction between Wzz and Wzy, however evidence for this was only provided
recently. Here we describe the tandem affinity purification and preliminary results of
the ongoing structural characterisation of Wzz with Wzy via cryo-EM. Not only the
final O-antigen chain length is serotype-specific, but also the components of the Oantigen
repeat unit. Modified heptoses present in the capsule of C. jejuni and Y.
pseudotuberculosis have been shown to play a key role in virulence. Here we describe
the structures of the C6 dehydratase DmhA, required for 6-deoxyheptose biosynthesis in
Y. pseudotuberculosis O:2a, the C5/C3 epimerase MlghB and the C4 reductase MlghC,
both required for O-methyl-L-gluco-heptose biosynthesis in C. jejuni strain NCTC
11168, and the C3 epimerase DdahB and the C4 reductase DdahC, both required for 6-
deoxy-D-altro-heptose biosynthesis in C. jejuni strain 81-176. These structures and the
mechanisms we propose will improve our understanding of the biosynthesis of these
unique sugars.
Type
Thesis, PhD Doctor of Philosophy
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
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Description of related resources
Woodward, L. & Naismith, J. H. 2016. Bacterial polysaccharide synthesis and export. Current Opinion in Structural Biology, 40, 81-88
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