MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

Gracïa, A. P.; Nikitushkin, V.; Ellerhorst, M.; Vilhena, C.; Klassert, T.; Starick, A.; Siemers, M.; Al-Jammal, W. K.; Vilotijevic, I.; Slevogt, H.; Papenfort, K.; Lackner, G.

doi:10.1101/2024.03.26.586840

bioRxiv · 10.1101/2024.03.26.586840

MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

Gracïa, A. P.·Nikitushkin, V.·Ellerhorst, M.·Vilhena, C.·Klassert, T.·Starick, A.·Siemers, M.·Al-Jammal, W. K.·Vilotijevic, I.·Slevogt, H.·Papenfort, K.·Lackner, G.

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Abstract

Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria.. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the {Delta}mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

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Gracïa, A. P., Nikitushkin, V., Ellerhorst, M., Vilhena, C., Klassert, T., Starick, A., Siemers, M., Al-Jammal, W. K., Vilotijevic, I., Slevogt, H., Papenfort, K., Lackner, G.. 2024-03-28. MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration. https://doi.org/10.1101/2024.03.26.586840

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