Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface

Bioscience Reports, vol. 42

Abstract

Gut microbial production of trimethylamine (TMA) from l-carnitine is directly linked to cardiovascular disease. TMA formation is facilitated by carnitine monooxygenase, which was proposed as a target for the development of new cardioprotective compounds. Therefore, the molecular understanding of the two-component Rieske-type enzyme from Escherichia coli was intended. The redox cofactors of the reductase YeaX (FMN, plant-type [2Fe-2S] cluster) and of the oxygenase YeaW (Rieske-type [2Fe-2S] and mononuclear [Fe] center) were identified. Compounds meldonium and the garlic-derived molecule allicin were recently shown to suppress microbiota-dependent TMA formation. Based on two independent carnitine monooxygenase activity assays, enzyme inhibition by meldonium or allicin was demonstrated. Subsequently, the molecular interplay of the reductase YeaX and the oxygenase YeaW was addressed. Chimeric carnitine monooxygenase activity was efficiently reconstituted by combining YeaX (or YeaW) with the orthologous oxygenase CntA (or reductase CntB) from Acinetobacter baumannii. Partial conservation of the reductase/oxygenase docking interface was concluded. A structure guided mutagenesis approach was used to further investigate the interaction and electron transfer between YeaX and YeaW. Based on AlphaFold structure predictions, a total of 28 site-directed variants of YeaX and YeaW were kinetically analyzed. Functional relevance of YeaX residues Arg271, Lys313 and Asp320 was concluded. Concerning YeaW, a docking surface centered around residues Arg83, Lys104 and Lys117 was hypothesized. The presented results might contribute to the development of TMA-lowering strategies that could reduce the risk for cardiovascular disease.

Authors 10

1. Fabian Piskol

   Technische Universität Braunschweig

   Affiliation as printed

   1Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

2. Kerstin Neubauer

   Technische Universität Braunschweig

   Affiliation as printed

   1Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

3. M. Eggers

   Technische Universität Braunschweig

   Affiliation as printed

   1Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

4. Lisa Margarete Bode

   Technische Universität Braunschweig

   Affiliation as printed

   1Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

5. Jan Jasper

   Technische Universität Braunschweig

   Affiliation as printed

   1Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

6. Alan John Slusarenko Aachen 2Institut für Biologie III

   RWTH Aachen University

   Affiliation as printed

   2Institut für Biologie III, RWTH Aachen University, Aachen, Germany

7. Edward J. Reijerse

   Max Planck Institute for Chemical Energy Conversion

   Affiliation as printed

   3Max-Planck-Institute for Chemical Energy Conversion, Mülheim an der Ruhr, Germany

8. Wolfgang Lubitz

   Max Planck Institute for Chemical Energy Conversion

   Affiliation as printed

   3Max-Planck-Institute for Chemical Energy Conversion, Mülheim an der Ruhr, Germany

9. Dieter Jahn

   Technische Universität Braunschweig

   Affiliation as printed

   4Braunschweig Centre of Integrated Systems Biology, Braunschweig, Germany

10. Jürgen Moser corresponding

    Technische Universität Braunschweig

    Affiliation as printed

    1Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany