CAREER: Defining the chemical contributions of Val29 and Tyr30 in Mycofactocin Biosynthesis for the Development of Novel Redox Molecules.

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With the support from the Chemistry of Life Processes (CLP) Program in the Division of Chemistry (CHE), Dr. John A. Latham from the University of Denver will investigate how the chemistry is carried out in the synthesis of the bacterial vitamin, mycofactocin. Bacteria produce numerous compounds, or natural products that enable competition for resources, detoxifying environments, and carrying out critical cellular processes. Mycofactocin, a bacterial vitamin which is required for shuttling electrons in critical cellular processes in Mycobacteria. Mycofactocin is made from a peptide using a powerful enzyme, MftC, in a way that is unusual in comparison to other peptide natural products. This study is expected to result in new understanding of this chemistry and a new class of bioengineered redox cofactors, which in turn will help to elucidate mycofactocin-dependent physiological processes. This work allows for graduate students and middle school science teachers to gain specialized training in mechanistic enzymology and rational bioengineering. In addition, through outreach programming, this work will incorporate products produced by high school and middle school students. Mycofactocin maturase, MftC, is a radical S-adenosylmethionine enzyme (rSAM) that catalyzes the most critical steps in mycofactocin biosynthesis. It is just one of a broad class of radical SAM enzymes in Nature and so mechanistic insights gleaned in this project will likely have much broader scientific impact upon studies and understanding of other members of this important enzymatic class. MftC modifies the precursor peptide, MftA, by oxidatively decarboxylating the C-terminal tyrosine and by the formation of a lactam between the penultimate valine and the C-terminal tyrosine. The reaction scope of MftC will be evaluated to determine the requirement of an electron sink (tyrosine) on the C-terminus and if the conservation of valine tunes the midpoint potential of mycofactocin. Objective 1 focuses on defining the mechanistic role of tyrosine by evaluating kinetic isotope effects and pKa effects of tyrosine analogs. Objective 2 establishes the chemical logic for the conservation of valine by evaluating substituent effects on the midpoint potential of mycofactocin. The project will advance the understanding rSAM mechanisms and establish the framework for the rational design of a new class of redox cofactors.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.