The Mechanism of Enzyme-catalyzed Ergothioneine Degradation Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Alice Maurer aus Deutschland Basel, 2020 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Florian Seebeck Prof. Dr. Michael Müller Basel, den 15.09.2020 Prof. Dr. Martin Spiess Dekan der Philosophisch-Naturwissenschaftlichen Fakultät Abstract The sulfur containing histidine derivative ergothioneine is a ubiquitous natural product. Research on its biosynthesis and degradation can elucidate the complex biological and molecular function of this small molecular weight compound. The biosynthesis of ergothioneine is well established, yet little is known about its degradation. The first step of ergothioneine degradation is catalyzed by the enzyme ergothionase, which will be the focus of this thesis. Ergothionase catalyzes the 1,2-elimination of trimethylamine from ergothioneine to yield thiourocanic acid. In this work, kinetic and structural investigations elucidate the mechanism of ergothionase. Based on the identification of catalytic residues, we are able to portray ergothionase producing organisms and found that they are mainly gut bacteria. This finding is in particular interesting because ergothioneine as food-additive is generally regarded as safe, whereas the ergothionase-mediated degrading of ergothioneine yields to trimethylamine, which is toxic. Furthermore, we characterized two unknown lyases. One of these new lyases employs a similar mechanism as ergothionase but uses an oxidized substrate derivative. Whereas, the other new lyase catalyzes the elimination of trimethylamine from trimethylhistidine (TMH) and has distinctive differences in the active site compared to ergothionase. The function and position of the catalytic acid in the active site of TMH-lyase suggest a mechanistic variation of the thought to be well-known 4- methylideneimidazol-5-one (MIO) dependent aromatic amino acid lyases: maybe the posttranslational formed MIO-moiety does not only serve as electron sink but also as catalytic acid. Studies on the phylogeny of ergothionase, TMH-lyase, aromatic amino acid lyase and the aspartase/fumarase superfamily propose that ergothionase has evolved prior to the aromatic amino acid lyases. The long evolutionary history of ergothionase underscores ergothioneine as ancient molecule. However, it is questioning that on the one hand an aromatic amino acid lyase represents the first step of the ubiquitous histidine degradation pathway, whereas on the other hand histidine builds the basis for ergothioneine biosynthesis. This finding might suggest the presence of an unknown alternative histidine degradation pathway. In addition, we have not only studied the degradation but also the biosynthesis of ergothioneine. Thereby, we focused on the regulation of EgtD, the methyltransferase of ergothioneine biosynthesis, and showed that this enzyme is not subject of phosphorylation in vitro. i Table of Contents Abstract .................................................................................................................................................... i Abbreviations .......................................................................................................................................... iv 1 Introduction ..................................................................................................................................... 1 1.1 Diversity of Ammonia-Lyases .................................................................................................. 1 1.1.1 Aspartase/Fumarase Superfamily ................................................................................... 3 1.1.2 Methylaspartate Ammonia-Lyase ................................................................................... 4 1.1.3 Aminoacyl-CoA Ammonia-Lyase...................................................................................... 6 1.1.4 Hydroxy Amino Acid Dehydratase/Deaminase ............................................................... 7 1.1.5 Ethanolamine Ammonia-Lyase ........................................................................................ 9 1.1.6 Amino Acid Cyclodeaminase ......................................................................................... 10 1.1.7 Aromatic Amino Acid Ammonia-Lyase .......................................................................... 12 1.2 Application in Biocatalysis of Ammonia-Lyases .................................................................... 15 1.3 Histidine Degradation ............................................................................................................ 17 1.4 Ergothioneine ........................................................................................................................ 19 1.4.1 Biosynthesis of Ergothioneine ....................................................................................... 20 1.4.2 Degradation of Ergothioneine ....................................................................................... 22 2 Aim of this Thesis........................................................................................................................... 24 3 The Mechanism of Ergothionase ................................................................................................... 25 3.1 Selection of a Specific Enzyme for Kinetic and Structural Characterization ......................... 26 3.2 Ergothionase Activity: Acid-Base Catalysis ............................................................................ 27 3.2.1 Activity of Ergothionase in Dependence of pH ............................................................. 28 3.2.2 Crystal Structure ............................................................................................................ 29 3.2.3 Identification of Important Catalytic Residues: Activity of Mutants ............................. 31 3.2.4 Function of Lys64 ........................................................................................................... 34 3.3 Substrate Activation .............................................................................................................. 36 3.3.1 Substrate Specificity ...................................................................................................... 38 3.3.2 Substrate Specificity of the K384M Variant .................................................................. 40 3.3.3 Ergothioneine Sulfonic Acid as Substrate ...................................................................... 42 3.3.4 Impact of Desmethyl-Ergothioneine ............................................................................. 44 3.3.5 Analysis of the Substrate and Solvent Isotope Effect of kcat.......................................... 45 3.4 Irreversible Substrate Binding Mechanism ........................................................................... 48 3.4.1 Substrate Isotope Effect Reveals Partial Irreversible Substrate Binding ....................... 49 3.4.2 Destabilization of the Closed Loop Formation .............................................................. 51 ii 3.4.3 Impact of the N63C Variant ........................................................................................... 52 3.5 Occurrence of Ergothionase .................................................................................................. 55 3.6 Identification of a New Ergothioneine Sulfonic Acid Lyase ................................................... 55 3.7 Conclusion ............................................................................................................................. 58 3.8 Experimental ......................................................................................................................... 59 4 TMH-Lyase ..................................................................................................................................... 97 4.1 Identification of a TMH-Lyase ............................................................................................... 97 4.2 Crystal Structure .................................................................................................................... 99 4.3 Kinetic Investigations of the Wild Type ............................................................................... 100 4.3.1 Substrate Specificity .................................................................................................... 100 4.3.2 pH-Dependence ........................................................................................................... 102 4.3.3 Substrate and Solvent Isotope Effects ......................................................................... 102 4.4 Important Catalytic Residues of TMH-Lyase Compared to Ergothionase and Histidine Ammonia-Lyase ............................................................................................................................... 103 4.4.1 Implications on the Mechanism of MIO-dependent Enzymes .................................... 107 4.5 Important Residues for MIO-Formation in Histidine Ammonia-Lyase ................................ 109 4.6 Phylogenetic Development ................................................................................................. 111 4.7 Influence