Unravelling the Mechanism of Fdc1, a Novel Prfmn Dependent Decarboxylase
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Unravelling the Mechanism of Fdc1, a Novel prFMN Dependent Decarboxylase A thesis submitted to the University of Manchester for the degree of Doctor in Philosophy (PhD) in the Faculty of Science and Engineering School of Chemistry Samuel S. Bailey 2018 Table of Contents 1 Abstract and Preface 0 1.1 Abstract 0 1.2 Declaration 1 1.3 Copyright Statement 1 1.4 Acknowledgments 2 1.5 Preface to Journal Format 3 2 Introduction 5 2.1 Project Outline 5 2.2 Biofuels 5 2.2.1 Second generation biofuels 6 2.3 Flavoenzymes 7 2.3.1 Covalent Modifications of Flavins 9 2.3.2 The Reactions of Flavin with Oxygen 10 2.4 Diels-Alderases: [4+2] Cycloaddition in Natural Product Biosynthesis 12 2.4.1 Artificial Diels-Alderases 13 2.4.2 Natural Diels-Alderases 13 2.5 The Role of Substrate Strain in Enzymatic Catalysis 14 2.5.1 Substrate Strain in Chelatases 15 2.5.2 Substrate Strain in Lysozyme 15 2.5.3 Substrate Strain in Human Transketolase 16 2.6 Enzymatic Decarboxylation 18 2.6.1 Cofactor Independent Decarboxylases 18 2.6.2 Thiamine Pyrophosphate (TPP) Dependent Decarboxylases 20 2.6.3 Pyridoxal 5’-phosphate (PLP) Dependent Decarboxylases 20 2.6.4 Flavin dependent decarboxylases 21 2.7 The UbiX/UbiD and Pad/Fdc systems 22 2.7.1 UbiX is responsible for the synthesis of a novel flavin cofactor 23 2.7.2 Fdc1 uses prFMN for decarboxylation 26 2.7.3 The structure of A. niger Fdc1 27 2.7.4 Proposed Mechanism of Decarboxylation by Fdc1 27 2.8 Other UbiD enzymes 29 1 2.8.1 The UbiD Superfamily 29 2.8.2 Common themes among the UbiD superfamily 31 2.9 Unravelling the UbiD mechanism: Fdc1 as a model system 31 2.9.1 Fdc1 Cofactor maturation 31 2.9.2 Mechanism of Decarboxylation 32 2.9.3 The ERE motif 32 2.10 Aims 33 2.11 References 33 3 The role of conserved residues in UbiD/Fdc decarboxylase in oxidative maturation, isomerisation and catalysis of prenylated flavin mononucleotide 46 3.1 Abstract 47 3.2 Introduction 47 3.3 Results 50 3.3.1 Light-dependent cofactor isomerization and enzyme inactivation 50 3.3.2 UV-vis spectrophotometric characterization of Fdc1 E277 and E282 variants reveal minor variation in prFMN incorporation and maturation 52 3.3.3 An acidic residue at both position 277 and 282 is required for activity 54 3.3.4 Enzyme catalysed styrene H/D exchange confirms the need for acidic residues at 277 and 282 55 3.3.5 Light-dependent cofactor isomerization is not dependent on the E-R- E motif 58 3.3.6 R173 variants reveal deficiencies in cofactor maturation 58 3.3.7 Crystal structure of R173A confirms cofactor maturation occurs over longer timescales 61 3.3.8 Crystal structures of Fdc1 E282 variants reveal minor structural variation 62 3.3.9 Crystal structures of Fdc1 E277 variants reveal significant C1’ adduct formation 63 3.3.10 Similar trends are observed for Saccharomyces cerevisiae Fdc1 variants 64 2 3.4 Discussion 67 3.5 Experimental Procedures 71 3.5.1 Cloning 71 3.5.2 Mutagenesis 71 3.5.3 Protein expression 71 3.5.4 Purification of A. niger Fdc1 71 3.5.5 Purification of S. cerevisiae Fdc1 72 3.5.6 In vitro reconstitution of apo-Fdc1 72 3.5.7 UV-visible spectroscopy and protein quantification 72 3.5.8 UV-visible spectrophotometric decarboxylation assays 72 3.5.9 Light/dark studies 73 3.5.10 Hydrogen/Deuterium exchange assays 73 3.5.11 HPLC decarboxylation assays 73 3.5.12 Protein crystallization and structure determination 73 3.5.13 EPR and ENDOR spectroscopy 76 3.5.14 Mass Spectrometry 76 3.6 References 76 4 Atomic description of an enzyme reaction dependent on reversible 1,3-dipolar cycloaddition 80 4.1 Abstract 81 4.2 Introduction 81 4.3 Results and Discussion 83 4.4 Methods 95 4.4.1 Cloning 95 4.4.2 Mutagenesis 95 4.4.3 Protein Expression 95 4.4.4 Protein Purification 95 4.4.5 UV-vis spectroscopy 96 4.4.6 UV-vis decarboxylation assays 96 4.4.7 Stopped-flow 96 4.4.8 Protein crystallization and structure determination 97 4.4.9 Production and crystallisation of FMN containing A. niger Fdc1 99 3 4.4.10 Crystallisation of A. niger Fdc1 containing prFMN-crotonic acid adduct 99 4.4.11 HPLC decarboxylation assays 99 4.4.12 Cofactor Mass Spectrometry 100 4.4.13 Protein Mass Spectrometry 100 4.5 References 100 5 Discussion 104 5.1 prFMNiminium is the active form of the cofactor 104 5.2 The mechanism of prFMN oxidative maturation 105 5.3 The mechanism of Decarboxylation by Fdc1 106 5.4 Outstanding Prospects in UbiD Biochemistry 110 5.5 Final remarks 112 5.6 References 112 6 Appendix 117 6.1 Published Paper: “The role of conserved residues in UbiD/Fdc decarboxylase in oxidative maturation, isomerisation and catalysis of prenylated flavin mononucleotide” 117 6.2 List of abbreviations 134 4 Table of figures and tables Chapter 2 Figure 1. General flavochemistry 8 Figure 2. General scheme for a diels-alderase catalysed [4+2] cycloaddition 12 Figure 3. The effect of enzymatic strain on the energy of the enzyme substrate and enzyme-transition state complex 14 Figure 4. Mesoporphyrin ring 15 Figure 5. A representation of a NAM sugar residue in the chair (a) and sofa (b) conformations 16 Figure 6. Crystal structure of the covalent D-xyulose-5-phosphate (X5P)-TDP intermediate in Transkelotase 18 Figure 7. Cofactor Independent Decarboxylations 19 Figure 8. A general mechanism for Thiamine Pyrophosphate (TPP) dependent decarboxylation 20 Figure 9. A general mechanism for Pyridoxal 5’-phosphate (PLP) dependent decarboxylation 21 Figure 10. A general mechanism for FMN dependent decarboxylation 22 Figure 11. UbiX overview 24 Figure 12. Fdc1 overview 26 Chapter 3 Figure 1. Overview of prFMN maturation and catalysis in Fdc1 48 Figure 2. Effect of illumination on A. niger Fdc1 activity 51 Figure 3. UV-visible spectra for A. niger Fdc1 variants 52 Figure 4. prFMN mass spectrometry and maturation mechanism 53 Figure 5. Detection of decarboxylation activity using HPLC 54 Figure 6. Characterization of the active variants E282D and E277D 55 Figure 7. H/D exchange of styrene 57 Figure 8. UV-induced tautomerisation of prFMNiminium to prFMNketimine 58 Figure 9. UV-Visible absorbance, EPR and ENDOR spectra of prFMN- reconstituted Fdc1 R173A and as purified Fdc1 R173A 60 Figure 10. Crystal structures of Fdc1 variants in complex with prFMN 62 0 Figure 11. UV-visible spectra and kinetic analysis for S. cerevisiae Fdc1 mutants 65 Figure 12. Crystal structures of S. cerevisiae Fdc1 variants in complex with prFMN 66 Figure 13. Proposed mechanism for maturation of prFMNreduced to prFMNiminium in Fdc1 69 Chapter 4 Figure 1. A. niger Fdc proposed enzyme mechanism 82 Figure 2. Crystal structures of A. niger Fdc with alkene substrates 83 Figure 3. Crystal structures of wild-type A. niger Fdc with various substrate analogues 84 Figure 4. UV-Visible spectra for A. niger with substrate analogues and HPLC activity assay 85 Figure 5. Stopped-flow traces and concentration profiles for A. niger Fdc1 with substrate analogues 86 Figure 6. Crystal structures of A. niger Fdc with alkyn substrate analogues 87 Figure 7. Mass spectrometry of prFMN-cycloadducts extracted from A. niger Fdc1 88 Figure 8. Solution studies and crystal structures for A. niger Fdc1 variants 90 Figure 9. A schematic overview illustrating the role of strain in the Fdc reaction 93 Chapter 5 Figure 1. The expanding prFMN chemical repertoire 105 Figure 2. Overview of the UbiD enzyme family 109 1 1 Abstract and Preface 1.1 Abstract The UbiD superfamily of enzymes are reversible decarboxylases that depend on the novel cofactor prenylated FMN (prFMN) for activity. The partner protein UbiX prenylates FMNH2 using dimethylallyl phosphate. The UbiX product, prFMNH2 is then passed to UbiD, where oxidative maturation to an active form, prFMNiminium occurs. Following oxidative activation, UbiD family members are able to catalyse the reversible decarboxylation of a wide range of unsaturated aliphatic and aromatic acids. The UbiD family contain a conserved active site E(D)RE motif that is proposed to play a role in cofactor maturation and decarboxylation. The Fdc1 enzyme from Aspergillus niger catalyses the decarboxylation of cinnamic acid and acts as a model system for studying UbiD enzymes, due to its amenability to high resolution crystallographic studies, ease of purification and readily detectable decarboxylation activity. Our studies on Fdc1 provide valuable insights into the role of the conserved ERE motif in cofactor maturation, isomerisation, and substrate (de)carboxylation. We confirm prFMNiminium is the active form, with illumination leading to inactivation through formation of the prFMNketimine isomer. We demonstrate ERE predominantly acts in catalysis, but not maturation or isomerisation. Our crystallographic studies of Fdc1 in complex with alkene substrates and alkyne inhibitors provides the first direct evidence for enzymatic 1,3-dipolar cycloaddition. These crystal structures also demonstrate a crucial role for molecular strain, whereby the enzyme is able to achieve reversible cycloaddition through selective destabilisation of key intermediates. Hence, we present strong evidence for the proposed 1,3-dipolar cycloaddition mechanism in case of enoic acid substrates. This leads to the question whether a similar mechanism also applies in case of aromatic acids, as occurs in distinct branches of the UbiD family. This remains a contentious issue, and will require future studies on distinct representatives of the UbiD-superfamily.