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 O-antigen 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-ʟ-gluco-heptose biosynthesis in C. jejuni strain NCTC 11168, and the C3 epimerase DdahB and the C4 reductase DdahC, both required for 6-deoxy-ᴅ-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.

Date of Award	27 Jun 2018
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Jim Naismith (Supervisor)