Oral Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Natural and De Novo Designed bi-functional peroxidases (KatGs): the role of the [Fe(IV)=O Trp.] intermediates in the intrinsic isoniazid resistance of Mycobacterium abscessus KatG. (#530)

Angela S. Gehrckens 1 , Joseph Phillips 2 , Iago De Assis Modenez 3 , Stéphane Canaan 1 , Anna F. A. Peacock 2 , Vincent L. Pecoraro 3 , Anabella Ivancich 1
  1. Research Unit UMR 7281, CNRS & Aix-Marseille University, Marseille, France
  2. School of Chemistry, University of Birmingham, Birmingham, UK
  3. Department of Chemistry, University of Michigan, Ann Arbor, USA

The intrinsic resistance of Mycobacterium abscessus to the frontline prodrug isoniazid (INH) has been correlated with the lack of INH activation by the heme-containing bi-functional enzyme KatG. Sequence alignment of the KatGs from Mycobacterium tuberculosis (MtKatG) and Mycobacterium abscessus (MabsKatG) shows a sequence identity of approximately 72%. However, it also indicates that the most common naturally-occurring mutations in MtKatG, that confer resistance to INH treatment in clinical isolates, are absent in MabsKatG. In order to elucidate the molecular mechanism of the Mabs-intrinsic resistance, we have characterized the catalytically active intermediates of MabsKatG by EPR spectroscopy and compared them with those that we have previously identified in MtKatG1 and B. pseudomallei KatG2,3.

Initial results from our 9-GHz EPR studies of the high-valent intermediates in MabsKatG show the formation of an exchange-coupled Trp radical as well as another protein-based radical, the later seemingly being different to the Trp.136 and Trp.153 radicals (BpKatG numbering) previously identified2. In this context, the characterization of the reaction of MabsKatG with INH will be discussed.

Exploiting de novo metalloprotein design4, we are developing novel artificial catalysts with heme & Trp redox cofactors, mimicking the strategies used by natural catalysts/heme enzymes in intramolecular electron transfer and the formation of radical intermediates. Modified peptide sequences of self-assembling three-stranded coiled-coil scaffolds in complex with heme and the introduction of a site to stabilize a Trp radical formed subsequently to the (FeIV=O Por.+) intermediate, mimicking the protein-based radical chemistry of KatGs, will also be presented.

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  3. (3) Ivancich, A.; Donald, L.D.; Villanueva, J.; Wiseman, B.; Fita, I.; Loewen, P.C. Biochemistry 2013, 52, 7271−7282.
  4. (4) Koebke, K.J.; Kühl, T.; Lojou, E.; Demeler, B.; Schoepp-Cothenet, B.; Iranzo, O.; Pecoraro, V.L.; Ivancich, A. Angew. Chem. Int. Ed. 2021, 60, 3974-3978.
  5. (5) Phillips, J.; De Assis Modenez, I.; Harbort, J. S.; Schoepp-Cothenet, B.; Pecoraro*, V.L.; Peacock*, A. F.; Ivancich*, A. (in preparation).