Accessing Pseudaminic Acid (Pse5Ac7Ac) containing Glycosides through the Characterisation of Pse5Ac7Ac Processing Enzymes Emily Kate Pedley Flack PhD University of York Chemistry August 2019 Abstract Cell-surface carbohydrate pseudaminic acid (Pse5Ac7Ac) is known to contribute to the virulence of several multi-drug resistant bacterial pathogens.1 Pse5Ac7Ac and its derivatives are not commercially available in appreciable quantities and chemical synthesis of these molecules has proved to be challenging.2–9 Access to Pse5Ac7Ac and activated CMP-Pse5Ac7Ac has been a hindrance in studies into the biological significance of Pse5Ac7Ac, including Pse5Ac7Ac-processing enzymes, which may be novel therapeutic targets.1 This project aimed to characterise enzymes which process pseudaminic acid and to chemoenzymatically synthesise glycosides which contain pseudaminic acid. Firstly, nucleotide-activated pseudaminic acid (CMP-Pse5Ac7Ac) was produced via a chemoenzymatic synthesis route. Six recombinant biosynthetic enzymes which are encoded in Campylobacter jejuni and Aeromonas caviae were purified for use in this reaction. Purification and characterisation of the resultant CMP-Pse5Ac7Ac confirmed the role of Aeromonas caviae PseF as an α-CMP-Pse5Ac7Ac synthetase. With CMP-Pse5Ac7Ac in-hand, a library of bacterial sialyltransferases were assayed for activity with CMP-Pse5Ac7Ac as donor. Success from this initial screen led to the synthesis of glycosides containing β-linked Pse5Ac7Ac, mediated by promiscuous sialyltransferases. Efforts were made to recombinantly produce five putative glycosyltransferases which were predicted to use CMP-Pse5Ac7Ac or a derivative as their natural donor (pseudaminyltransferases) however, all proteins were initially insoluble. Acinetobacter baumannii retaining pseudaminyltransferase was solubilised through the construction of an Im9-fusion protein. Activity studies monitored by Liquid Chromatography – Mass Spectrometry confirmed that Im9-KpsS1 could utilise CMP-Pse5Ac7Ac as a donor. Finally, Im9-KpsS1 was used in a seven enzyme one-pot chemoenzymatic synthesis to produce α- 2,6-Pse5Ac7Ac-pNP-β-D-Glc, confirming that KpsS1 functions as a retaining pseudaminyltransferase. To our knowledge the work presented herein details the first examples of chemoenzymatic synthesis of glycosides containing Pse5Ac7Ac and the first in vivo study of a pseudaminyltransferases to provide unequivocal functional characterisation of this novel class of enzyme. 2 Contents 1. Abstract ...................................................................................................................... 2 2. Contents ..................................................................................................................... 3 3. Acknowledgements ................................................................................................... 7 4. Author’s Declaration .................................................................................................. 9 5. Chapter 1: Introduction ........................................................................................... 10 1.1 Carbohydrates in Biology ................................................................................. 10 1.1.1 Carbohydrate Structures .................................................................................. 10 1.1.1 Biological Significance of Glycosylation and Glycans ....................................... 12 1.2 Glycosyltransferases .............................................................................................. 16 1.2.1 Glycosyltransferase classification .................................................................... 16 1.2.2 Glycosyltransferase folds ................................................................................. 17 1.2.3 Glycosyltransferase function ............................................................................ 17 1.2.3 Mechanisms of glycosyltransferases ................................................................ 18 1.3 Sialic Acids ....................................................................................................... 23 1.3.1 Sialic Acid Structures and Function in Eukaryotes ........................................ 23 1.3.2 Sialic acid biosynthesis and scavenging in bacteria ......................................... 24 1.3.3 Sialic acid in bacterial glycoconjugates ............................................................ 25 1.4 Pseudaminic Acids ................................................................................................. 26 1.4.1 Occurrence and Biological Significance of Pseudaminic Acids ........................ 26 1.4.2 Biosynthesis of Nucleotide-Activated Pseudaminic Acids ................................ 28 1.4.3 Chemical synthesis towards Pseudaminic Acids and Pseudaminic Acid glycosides ................................................................................................................. 33 1.4.4 Chemoenzymatic synthesis of Pseudaminic Acids ........................................... 44 1.4.4 Introduction to Pseudaminic Acid Processing Enzymes ................................... 47 1.5 Project Outline ........................................................................................................ 49 6. Chapter 2: Chemoenzymatic Synthesis of CMP-Pseudaminic Acid ..................... 50 3 2.1 Introduction ............................................................................................................ 50 2.2 Experimental .......................................................................................................... 54 2.2.1 Overxpression and Purification of PseB, PseC, PseH, PseG and PseI ............ 54 2.2.2 Enzymatic synthesis of Pse5Ac7Ac with Acetyl-thiocholine Iodide ................... 56 2.2.3 Small-molecule screen for potential inhibition of PseB and PseI ...................... 56 2.2.4 Recombinant Expression and purification attempt of C. jejuni PseF in E. coli .. 58 2.2.5 Expression and purification of A. caviae PseF ................................................. 60 2.2.6 Activity assay of A. caviae PseF with Pse5Ac7Ac ............................................ 61 2.2.7 Purification of Aeromonas caviae PseF for Crystallisation trials ....................... 61 2.2.8 A. caviae PseF Protein Identification by Mass Spectrometry ........................... 62 2.2.9 A. caviae PseF Circular Dichorism ................................................................... 62 2.2.10 A. caviae PseF Size Exclusion Chromatography - Multi-Angle Laser Light Scattering ................................................................................................................. 63 2.2.11 Crystalisation trials for A. caviae PseF ........................................................... 63 2.2.12 Large scale synthesis and purification of CMP-Pse5Ac7Ac, via a one-pot six enzyme synthesis ..................................................................................................... 64 2.3 Results ................................................................................................................... 66 2.3.1 Overxpression and Purification of PseB, PseC, PseH, PseG and PseI ............ 66 2.2.3 Enzymatic synthesis of Pse5Ac7Ac with Acetyl-thiocholine Iodide ................... 67 2.3.3 Small-molecule screen for potential inhibition of PseB and PseI ...................... 68 2.3.4 Recombinant Expression and purification attempt of C. jejuni PseF in E. coli .. 72 2.3.5 Expression and purification of A. caviae PseF ................................................. 73 2.3.6 Activity assay of A. caviae PseF with Pse5Ac7Ac ............................................ 74 2.3.7 Purification of A. caviae PseF for Crystallisation trials ...................................... 76 2.3.8 A. caviae PseF Protein Identification by Mass Spectrometry ............................ 77 2.3.9 A. caviae PseF Circular Dichorism ................................................................... 78 2.3.10 A. caviae PseF Size Exclusion Chromatography - Multi-Angle Laser Light Scattering ................................................................................................................. 79 2.3.11 Crystallisation trials for Aeromonas caviae PseF ........................................... 79 4 2.3.12 Large scale synthesis and purification of CMP-Pse5Ac7Ac, via a one-pot six enzyme synthesis ..................................................................................................... 80 2.4 Discussion .............................................................................................................. 83 2.5 Conclusions and Future Work ................................................................................ 90 7. Chapter 3: Sialyltransferase mediated synthesis of glycosides containing Pseudaminic Acid .................................................................................................... 91 3.1 Introduction ............................................................................................................ 91 3.2 Experimental .......................................................................................................... 94 3.2.1 Initial Activity assays of sialyltransferase library with CMP-Pse5Ac7Ac donor .. 94 3.2.2 PmST acceptor screen with CMP-Pse5Ac7Ac as donor .................................. 96 3.2.3 Large Scale synthesis of β-Pse5Ac7Ac-2,3-pNP-β-ᴅ-Galp using PmST .......... 96 3.3 Results ..................................................................................................................