Functional models for iron(II)/α-keto acid dependent enzymes

Jonasson, Niko Sebastian Willem (2021): Functional models for iron(II)/α-keto acid dependent enzymes: a combined synthetic and analytical approach to elucidate the mechanisms of TET and HPDL enzymes. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Iron and iron enzymes are ubiquitous in nature. Besides heme-type enzymes, to which the well-known cytochrome P450 belongs, the superfamily of iron(II)/α-keto acid dependent enzymes is of utmost biological relevance. Enzymes of this class are involved in a number of important biochemical transformations including the synthesis of penicillin, the metabolism of taurine, DNA repair, or epigenetically relevant demethylation of DNA. The mechanism of action of these enzymes has been the focus of scientific investigations since 1982,[1] however, details of specific enzymatic transformations still remain elusive. In the years 2003-2005 a series of bioinorganic investigations, in part using synthetic model complexes, led to the discovery of an iron(IV)-oxido moiety as the active species in the enzymes’ catalytic cycle.[2–6] In 2009 ten-eleven translocation 5-methyl cytosine dioxygenase (TET) enzymes, members of the iron(II)/α-keto acid dependent enzyme superfamily, were discovered to play an integral part in the molecular processes of epigenetics by oxidizing the methyl group on the important epigenetic marker 5-methyl cytosine (5mC).[7] Whereas several biochemical investigations gave insight into the reactivity of TET enzymes, details are still unclear that warrant more thorough studies. For example, the reactivity of TET towards 5mC and its oxidized metabolites 5-hydroxymethyl cytosine (5hmC) and 5-formyl cytosine (5fC) unexpectedly does not correspond to the trend observed in the theoretically calculated bond dissociation energies (BDEs) of these substrates.[8] On another note, a defect in the gene coding for another iron(II)/α-keto acid dependent enzyme (4 hydroxyphenylpyruvate dioxygenase like, HPDL) was recently identified as the main cause for a neurodegenerative disorder.[9–11] Sequence, location, and some of the enzyme’s biochemical behavior including its substrate have been identified. However, there is reason to believe that not all intermediates have been identified unequivocally – not mentioning the final product nor the enzymes’ exact mechanism. Therefore, the goal of this work was to synthesize, study, and modify synthetic, iron-based model complexes for the study of the aforementioned enzymes. Once obtained, these functional models were then to be applied in detailed investigations with (model) substrates of both TET and HPDL. Expansion of the functional model complex platform in order to gain a more diverse portfolio of such iron-based model complexes represents an additional goal. The literature-known complex [FeIV(O)(Py5Me2H)]2+ (C-6; Py5Me2H = 2,6-bis(1,1-bis(2-pyridyl)ethyl)pyridine, L-1) is the basis of the functional model complex platform developed within the scope of this work.[12] Reaction with the nucleobase 5mC gave the expected TET metabolites 5hmC, 5fC, and 5-carboxy cytosine (5caC), as shown by GC-MS analysis. The mechanism of this reaction was elucidated by UV-vis kinetic investigations which allowed for the determination of the rate law as well as the identification of the rate-limiting step, hydrogen atom transfer (HAT) by the iron(IV)-oxido moiety from the methyl group of the substrate (chapter III.4).[13] Subsequently, the substrate’s complexity was increased to nucleosides (chapter III.5) and finally short oligonucleotides (10-mer). In a collaboration with the Carell research group at LMU Munich within the SFB1309 “Chemical Biology of Epigenetic Modifications” it was shown that C-6 is indeed capable of selectively oxidizing a 5mC residue within an oligonucleotide context. Side-product analysis was performed by MALDI-MS (matrix assissted laser desorption/ionization-mass spectrometry), which showed the formation of small amounts of end-of-strand decomposition, however, no interior strand breaks were observed (chapter III.7).[14] These findings lay the ground-work for ongoing investigations of the application of C-6 in epigenetic sequencing of DNA. The TET project was complemented by a joint SFB1309 investigation of the behavior of C-6 towards a series of natural and non-natural nucleobase substrates in collaboration with the Zipse research group at LMU Munich. Here, it was shown that BDEs are a good predictor for the reactivity of the functional model complex C-6 towards C-H bonds, particularly in a nucleobase context (chapter III.8).[15] Furthermore, the iron(III)-hydroxido complex [FeIII(OH)(L-1)]2+ (C-4) was successfully identified as the intermediate and product in the reaction of C-6 with organic substrates. Subsequent investigation of C 4 gave first insight into its reactivity in rebound reactions (chapter IV). In addition to these investigations on the application of C-6, the platform of iron-based functional model complexes was expanded: the ligand system Py5Me2H was modified at several locations, initial experiments towards the immobilization of C 6 on solid supports were successful, and a series of new iron(IV)-oxido, iron(III)-hydroxido, and iron(II) complexes were synthesized (chapter V). The findings presented in this work will give multiple opportunities to further continue on the bioinorganic analysis of iron(II)/α-keto acid dependent enzymes. Metabolomic studies on biological samples obtained from E. coli cultures overexpressing HPDL as well as mammalian cell cultures shed light on the substrate consumption, product formation, and the mechanism of HPDL. Model complex studies with C-6, its iron(III)-hydroxido derivative C-4, and several other iron compounds gave further insight into the complex interaction of HPDL with its substrates and intermediates (chapter VI).

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 11. November 2021
• 1. Bericht­erstatter:in: Daumann, Lena