DESIGN AND SYNTHESIS OF CHEMICAL TOOLS FOR STUDIES OF CARBOHYDRATE ACTIVE ENZYMES Marija Petricevic Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy October 2018 School of Chemistry The University of Melbourne ABSTRACT Glycoside hydrolases (GH) are enzymes that catalyse the hydrolysis of glycosidic linkages. They are classified into over 150 families based on their primary amino acid sequence. Family GH99 endo-α-1,2-mannosidases/endo-α-1,2-mannases cleave α- Glc/Man-1,3-α-Man-OR structures within mammalian N-linked glycans and fungal α- mannans. They are predicted to perform base-catalysed hydrolysis via an 1,2-anhydro sugar intermediate. The first part of this thesis reports the synthesis of a mechanism- inspired inhibitor, α-mannosyl-1,3-noeuromycin (ManNOE). ManNOE was found to be the most potent GH99 inhibitor to date. The success of ManNOE was attributed to a favourable interaction between the 2-OH on the NOE ring and active site residue E333, predicted to be important in mechanism. Also described is the synthesis of the inhibitor α-mannosyl-1,3-(2-amino)deoxymannojirimycin (Man2NH2DMJ). Modest affinities were observed for Man2NH2DMJ. Structural studies revealed it binds in the same way as other iminosugar inhibitors, suggesting poor inhibition is not due to failure of the 2-NH2 to bind to reside E333, but rather a reduction in basicity of the endocyclic nitrogen in the presence 2-NH2 protonation. The second part of this thesis examines a novel family GH134 β-1,4-mannanases. A GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. was found to possess a fold closely related to that of hen egg white lysozyme (HEWL) and to act with inversion of stereochemistry. X-ray crystallography and ab initio quantum mechanics (QM)/molecular mechanics (MM) metadynamics reveal this enzyme utilizes a unique 1 3 ‡ 3 C4→ H4 → H1 conformational itinerary along the reaction co-ordinate, different to any known β-1,4-mannanases. i The third part of this thesis examines sulfoglycolysis. Sulfoquinovosyl glycerol (SQGro) was synthesised as a mixture of diastereomers. A family GH31 sulfoquinovosidase (YihQ) isolated Escherichia coli (E. coli) was determined to have a 6-fold preference for the naturally occurring isomer (2’R-SQGro) but could cleave both isomers. SQGro supports growth of E. coli to find cell densities comparable to growth on glucose. Preliminary studies revealed that the plant pathogen Agrobacterium tumefaciens performs sulfoglycolysis when grown on SQ as the sole carbon source. Growth of cultures and consumption of SQ were directly correlated to release sulfite into the media, which over time autooxidised to sulfate. Proteomics enabled the discovery of the sulfoglycolysis gene cluster in A. tumefaciens which enabled prediction of a novel sulfoglycolytic pathway. ii DECLARATION This is to certify that: i. the thesis comprises only my original work towards the PhD except where indicated in the Preface, ii. due acknowledgement has been made in the text to all other material used, iii. the thesis is less than 100 000 words in length, exclusive of tables, bibliographies and appendices. Marija Petricevic October 2018 iii PREFACE All the work reported herein has been conducted by the candidate, except where indicated. The inhibitors α-D-mannopyranosyl-1,3-(1,2-dideoxy)mannose (ManddMan), α-D- mannopyranosyl-1,3-glucal (ManGlucal) and α-D-mannopyranosyl-1,3-mannoimidazole (ManManIm) in Chapters 2 and 3 were synthesised by Pearl Fernandes in the laboratory of Prof Spencer Williams (University of Melbourne). In Chapter 5 synthesis of (S)-2,3- dihydroxypropane-1-sulfonate (DHPS) was synthesised by Janice Mui under the guidance and supervision of the candidate. X-ray crystallographic studies and isothermal titration calorimetry with B. thetaiotaomicron, B. xylanisolvens GH99 enzymes as described in Chapters 2 and 3 were conducted by Lukasz Sobala in the laboratory of Prof. Gideon Davies (University of York). Structural analysis of SsGH134 as described in Chapter 4 was performed by Dr Yi Jin in the laboratory of Prof. Gideon Davies (University of York). Dissociation constants for the binding of ManddMan and ManGlucal to BtGH99 and BxGH99 were determined by 2D NMR in the laboratory of Prof Jesus Jiminez-Barbaro (Ikerbasque Basque Foundation for Science, Spain.) Classical molecular dynamics (MD) and QM/MM metadynamics described in Chapters 2 and 4 were conducted in the laboratory of Prof Carme Rovira with the assistance of Lluís Raich (Universitat de Bercelona, Spain). Expression plasmids in Chapter 4 were assembled by Alan John in the laboratory of Dr Ethan Goddard Borger (Walter and Eliza Hall Institute). Expression of proteins in Chapter 4 was conducted by the candidate with assistance of Alan John in the laboratory of Dr Ethan Goddard Borger. YihQ and AtSQase proteins in Chapters 5 and 6 were prepared by James Lingford in Dr Ethan Goddard-Borger's laboratory. Quantitative analysis of DHPS was performed with the assistance of Dr Eileen Ryan (University of Melbourne). A. tumefaciens C58 was a gift from Dr Monica Doblin (School of Botany, University of iv Melbourne). Proteomics in Chapter 6 was performed in collaboration with Dr Nicholas Scott (University of Melbourne). The work discussed in this thesis has been published in part: 1) ACS Central Science, 2018, 4, 1266-1273. Structural and Biochemical Insights into the Function and Evolution of Sulfoquinovosidases. 2) Chemistry - A European Journal, 2018, 29, 7464–7473. Exploration of strategies for mechanism-based inhibitor design for family GH99 endo-α- 1,2-mannanases. 3) Journal of the American Chemical Society, 2017, 139, 1089–1097. Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2- Mannanase. 4) ACS Central Science. 2016, 2, 896–903. A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism. Additional work, not included here, performed by the candidate: 1) Chem. Comm. 2016, 52, 11096-11099. Structural and mechanistic insights into a Bacteroides vulgatus retaining N-acetyl-β- galactosaminidase that uses neighbouring group participation. v ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Professor Spencer J. Williams. His enthusiasm for science is contagious and highly motivational. I cannot thank him enough for his invaluable support and guidance over the past few years. Thank you to Dr Ethan Goddard-Borger for introducing me to the world of biochemistry and extending my love of science in new directions. I gratefully acknowledge the funding sources that made this research possible. My PhD was funded by The Melbourne Research Scholarship and the Norma Hilda Schuster Scholarship. Thank you to all my colleagues for your friendship and help in the lab. You have made my PhD experience so much more enjoyable and memorable. I would like to thank my parents and sister, although they have no idea what I’ve been studying, their love and support throughout my many years of university has been limitless. A big thank you to my partner Charlie, who has been a constant source of moral support. Thanks for listening to me vent about all those failed experiments and accompanying me to check bacterial cultures at 3 am. I really appreciate it! vi TABLE OF CONTENTS ABSTRACT ...................................................................................................................... i DECLARATION ............................................................................................................. iii PREFACE ........................................................................................................................ iv ACKNOWLEDGEMENTS ............................................................................................ vi TABLE OF CONTENTS ............................................................................................... vii ABBREVIATIONS .......................................................................................................... x CHAPTER ONE: Introduction ......................................................................................... 1 1.1 Introduction to glycoside hydrolases ....................................................................... 2 1.2 Mechanisms of glycoside hydrolases ...................................................................... 3 1.3 Mechanism of hen egg white lysosome (HEWL) mechanism ................................ 4 1.4 Mechanism of goose egg white lysosome (GEWL) ................................................ 5 1.5 Neighbouring group participation (NGP) mechanism............................................. 6 1.6 Anti/Syn lateral protonation ..................................................................................... 8 1.7 Transition states of glycosidases ............................................................................. 9 1.8 Substrate distortion during enzymatic hydrolysis of glycosidases ........................ 12 1.9 Reaction co-ordinate .............................................................................................. 13 1.10 Scope of thesis ..................................................................................................... 20 CHAPTER TWO: Investigation of the effects of shape and charge on family GH99 endo-α-1,2-mannanases .................................................................................................. 22 2.1 N-Linked glycan biosynthesis in the secretory pathway ......................................