Mechanistic Insights into MftR-Dependent Regulation of the Redox Cofactor Mycofactocin

<p>Organic redox cofactors are essential for life. While classic flavins and nicotinamides are widely distributed across all domains of life, nature has also evolved niche cofactors in subsets of life domains. For example, in <em>Actinobacteria</em>, coenzyme F420 is commonly used in place of flavin mononucleotide in enzymes associated with carbon fixation and oxidation of secondary alcohols. The importance of niche cofactors has long been recognized however, detailed understanding about their biosynthesis and physiological uses has been lagging. One class of niche cofactors is derived from ribosomally synthesized and posttranslationally modified peptides (RiPPs). To achieve their mature form, the genetically encoded RiPP precursor peptide undergoes significant post-translational modifications by diverse families of tailoring enzymes. Following synthesis by the ribosome, modifying enzymes process the precursor peptide into the mature redox cofactor. Currently, there are two known RiPP-derived redox cofactors, pyrroloquinoline quinone, which has been well-characterized, and mycofactocin (MFT), which was recently discovered.</p> <p>While MFT biosynthetic proteins have been extensively characterized, the physiological conditions that require MFT is not well-understood. To gain insights into the regulation of MFT in <em>Mycobacterium smegmatis</em> mc2155, we investigated the DNA-binding and ligand-binding activity of the putative TetR-like regulator, MftR. Here, we demonstrated that MftR binds to the <em>mft</em> promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5’ to <em>mftA</em> and found it to be highly conserved in <em>M. tuberculosis, M. bovis, M. ulcerans,</em> and <em>M. marinum</em>. To determine when the <em>mft</em> biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoA’s with low micromolar affinities. To demonstrate that oleoyl-CoA induces the <em>mft</em> BGC <em>in vivo</em>, we re-engineered a fluorescent protein reporter system to express a MftA-mCherry fusion. Using the mCherry fluorescent readout, we show that the <em>mft</em> BGC is upregulated in<em> M. smegmatis</em> mc2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the <em>mft</em> BGC and that MFT production is induced by long chain acyl-CoA’s. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Year dc:date.available

2023

Author dc:creator

• Mendauletova, Aigera

Contributors dc:contributor

• Sandra S. Eaton
• Allegra Aron
• Michelle Knowles

• Organic redox cofactors
• Ribosomally synthesized and posttranslationally modified peptides (RiPPs)
• Mycofactocin (MFT)
• Biochemistry
• Biochemistry, Biophysics, and Structural Biology
• Chemistry
• Life Sciences
• Physical Sciences and Mathematics

Statement dc:rights

• <p>Copyright is held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

en

Repository record dc:identifier

https://digitalcommons.du.edu/etd/2222

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-3216