Mycofactocin is a putative ribosomally synthesized and post-translationally modified peptide (RiPP)-derived redox cofactor. Its biosynthesis is accomplished through the dedicated actions of the products of six conserved genes, mftABCDEF. The mycofactocin pathway is one of the most widely distributed RiPP systems in bacteria however, this distribution is heavily skewed towards the Mycobacteria genus including human pathogenic variants such as M. tuberculosis and M. ulcerans. Gene expression studies have demonstrated the essentiality of the pathway in the ability of M. tuberculosis to utilize the host's cholesterol as sole carbon source during latency. However, the biosynthesis, structure and physiological function of mycofactocin remain enigmatic. Current efforts to elucidate the biosynthesis, structure and function of mycofactocin have focused on in vitro reconstitution of each enzyme in the pathway to gain insights into their role and function.
The biosynthesis of mycofactocin commences with the ribosomal production of the precursor peptide MftA containing conserved C-terminal residues - IDGMCGVY. In the presence of the RRE domain MftB, the RS-SPASM enzyme MftC, catalyzes the SAM-dependent oxidative decarboxylation and carbon-carbon bond formation on MftA to form MftA*. The roles of the auxiliary [Fe-S] clusters in MftC catalysis as well as the subsequent steps in the biosynthesis of mycofactocin are not known. Here, we have provided additional information regarding the roles of the auxiliary clusters in MftC. We showed that MftC contains three [4Fe-4S] clusters, all of which are required for catalysis. In addition, we measured the midpoint potentials of the clusters to provide insights into the redox flipping mechanism of MftC. Furthermore, we reconstituted the activity of MftE and showed that it selectively hydrolyzes MftA* to form MftA (1-28) and a 3-amino-5-(4-hydroxybenzyl)-4,4-dimethylpyrrolidin-2-one, herein referred to as AHDP. From this study, we have clarified the misunderstandings surrounding the accurate precursor for mycofactocin biosynthesis. Subsequently, we reconstituted the activity of MftD and showed that it catalyzes the oxidative deamination of AHDP to form an α-keto moiety herein referred to as premycofactocin. Lastly, we measured the midpoint potential of premycofactocin to be ~ -255 mV and demonstrated that it is used by mycofactocin-associated short chain dehydrogenases for multiple catalytic turnover.
","abstract_html":"<p>Mycofactocin is a putative ribosomally synthesized and post-translationally modified peptide (RiPP)-derived redox cofactor. Its biosynthesis is accomplished through the dedicated actions of the products of six conserved genes, <em>mftABCDEF</em>. The mycofactocin pathway is one of the most widely distributed RiPP systems in bacteria however, this distribution is heavily skewed towards the <em>Mycobacteria</em> genus including human pathogenic variants such as <em>M. tuberculosis</em> <em>and M. ulcerans</em>. Gene expression studies have demonstrated the essentiality of the pathway in the ability of M. tuberculosis to utilize the host's cholesterol as sole carbon source during latency. However, the biosynthesis, structure and physiological function of mycofactocin remain enigmatic. Current efforts to elucidate the biosynthesis, structure and function of mycofactocin have focused on <em>in vitro</em> reconstitution of each enzyme in the pathway to gain insights into their role and function.</p> <p>The biosynthesis of mycofactocin commences with the ribosomal production of the precursor peptide MftA containing conserved C-terminal residues - IDGMCGVY. In the presence of the RRE domain MftB, the RS-SPASM enzyme MftC, catalyzes the SAM-dependent oxidative decarboxylation and carbon-carbon bond formation on MftA to form MftA*. The roles of the auxiliary [Fe-S] clusters in MftC catalysis as well as the subsequent steps in the biosynthesis of mycofactocin are not known. Here, we have provided additional information regarding the roles of the auxiliary clusters in MftC. We showed that MftC contains three [4Fe-4S] clusters, all of which are required for catalysis. In addition, we measured the midpoint potentials of the clusters to provide insights into the redox flipping mechanism of MftC. Furthermore, we reconstituted the activity of MftE and showed that it selectively hydrolyzes MftA* to form MftA (1-28) and a 3-amino-5-(4-hydroxybenzyl)-4,4-dimethylpyrrolidin-2-one, herein referred to as AHDP. From this study, we have clarified the misunderstandings surrounding the accurate precursor for mycofactocin biosynthesis. Subsequently, we reconstituted the activity of MftD and showed that it catalyzes the oxidative deamination of AHDP to form an α-keto moiety herein referred to as premycofactocin. Lastly, we measured the midpoint potential of premycofactocin to be ~ -255 mV and demonstrated that it is used by mycofactocin-associated short chain dehydrogenases for multiple catalytic turnover.</p>","abstract_has_math":false,"creators":["Ayikpoe, Richard Selorm"],"institution":null,"degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":null,"degree_department":null,"school":null,"contributors":["John A. Latham, Ph.D.","Martin Margiatti, Ph.D.","Michelle Knowles, Ph.D.","Brian Michel, Ph.D."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-01-01T08:00:00Z","date_published":"2019-01-01T08:00:00Z","updated_at":"2026-07-24T02:02:19Z","subjects":["Mossbauer","Iron-sulfur clusters","MftC","Mycofactocin","Oxidative deamination","Redox cofactor","Ribosomally synthesized","Post translationally modified peptide","Cell and Developmental Biology","Genetics and Genomics","Life Sciences"],"languages":["en"],"rights":["Copyright is held by the author. User is responsible for all copyright compliance.
"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.du.edu/etd/1645","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["John A. Latham, Ph.D.","Martin Margiatti, Ph.D.","Michelle Knowles, Ph.D.","Brian Michel, Ph.D."]},{"key":"dc:creator","label":"Author","values":["Ayikpoe, Richard Selorm"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2020-01-21T08:00:00Z"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Mossbauer","Iron-sulfur clusters","MftC","Mycofactocin","Oxidative deamination","Redox cofactor","Ribosomally synthesized","Post translationally modified peptide","Cell and Developmental Biology","Genetics and Genomics","Life Sciences"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright is held by the author. User is responsible for all copyright compliance.
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The biosynthesis of mycofactocin commences with the ribosomal production of the precursor peptide MftA containing conserved C-terminal residues - IDGMCGVY. In the presence of the RRE domain MftB, the RS-SPASM enzyme MftC, catalyzes the SAM-dependent oxidative decarboxylation and carbon-carbon bond formation on MftA to form MftA*. The roles of the auxiliary [Fe-S] clusters in MftC catalysis as well as the subsequent steps in the biosynthesis of mycofactocin are not known. Here, we have provided additional information regarding the roles of the auxiliary clusters in MftC. We showed that MftC contains three [4Fe-4S] clusters, all of which are required for catalysis. In addition, we measured the midpoint potentials of the clusters to provide insights into the redox flipping mechanism of MftC. Furthermore, we reconstituted the activity of MftE and showed that it selectively hydrolyzes MftA* to form MftA (1-28) and a 3-amino-5-(4-hydroxybenzyl)-4,4-dimethylpyrrolidin-2-one, herein referred to as AHDP. From this study, we have clarified the misunderstandings surrounding the accurate precursor for mycofactocin biosynthesis. Subsequently, we reconstituted the activity of MftD and showed that it catalyzes the oxidative deamination of AHDP to form an α-keto moiety herein referred to as premycofactocin. Lastly, we measured the midpoint potential of premycofactocin to be ~ -255 mV and demonstrated that it is used by mycofactocin-associated short chain dehydrogenases for multiple catalytic turnover.
"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["New Insights into Mycofactocin Biosynthesis, Structure and Function"]}]}],"canonical_facts":{"dc:contributor":["John A. Latham, Ph.D.","Martin Margiatti, Ph.D.","Michelle Knowles, Ph.D.","Brian Michel, Ph.D."],"dc:creator":["Ayikpoe, Richard Selorm"],"dc:date.available":["2020-01-21T08:00:00Z"],"dc:description.abstract":["Mycofactocin is a putative ribosomally synthesized and post-translationally modified peptide (RiPP)-derived redox cofactor. Its biosynthesis is accomplished through the dedicated actions of the products of six conserved genes, mftABCDEF. The mycofactocin pathway is one of the most widely distributed RiPP systems in bacteria however, this distribution is heavily skewed towards the Mycobacteria genus including human pathogenic variants such as M. tuberculosis and M. ulcerans. Gene expression studies have demonstrated the essentiality of the pathway in the ability of M. tuberculosis to utilize the host's cholesterol as sole carbon source during latency. However, the biosynthesis, structure and physiological function of mycofactocin remain enigmatic. Current efforts to elucidate the biosynthesis, structure and function of mycofactocin have focused on in vitro reconstitution of each enzyme in the pathway to gain insights into their role and function.
The biosynthesis of mycofactocin commences with the ribosomal production of the precursor peptide MftA containing conserved C-terminal residues - IDGMCGVY. In the presence of the RRE domain MftB, the RS-SPASM enzyme MftC, catalyzes the SAM-dependent oxidative decarboxylation and carbon-carbon bond formation on MftA to form MftA*. The roles of the auxiliary [Fe-S] clusters in MftC catalysis as well as the subsequent steps in the biosynthesis of mycofactocin are not known. Here, we have provided additional information regarding the roles of the auxiliary clusters in MftC. We showed that MftC contains three [4Fe-4S] clusters, all of which are required for catalysis. In addition, we measured the midpoint potentials of the clusters to provide insights into the redox flipping mechanism of MftC. Furthermore, we reconstituted the activity of MftE and showed that it selectively hydrolyzes MftA* to form MftA (1-28) and a 3-amino-5-(4-hydroxybenzyl)-4,4-dimethylpyrrolidin-2-one, herein referred to as AHDP. From this study, we have clarified the misunderstandings surrounding the accurate precursor for mycofactocin biosynthesis. Subsequently, we reconstituted the activity of MftD and showed that it catalyzes the oxidative deamination of AHDP to form an α-keto moiety herein referred to as premycofactocin. Lastly, we measured the midpoint potential of premycofactocin to be ~ -255 mV and demonstrated that it is used by mycofactocin-associated short chain dehydrogenases for multiple catalytic turnover.
"],"dc:format":["application/pdf"],"dc:identifier":["https://digitalcommons.du.edu/etd/1645"],"dc:language":["en"],"dc:rights":["Copyright is held by the author. User is responsible for all copyright compliance.
"],"dc:subject":["Mossbauer","Iron-sulfur clusters","MftC","Mycofactocin","Oxidative deamination","Redox cofactor","Ribosomally synthesized","Post translationally modified peptide","Cell and Developmental Biology","Genetics and Genomics","Life Sciences"],"dc:title":["New Insights into Mycofactocin Biosynthesis, Structure and Function"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-24T02:02:19Z"}