Defining intermediates and redundancies in coenzyme Q precursor biosynthesis.

Coenzyme Q (CoQ), a redox-active lipid essential for oxidative phosphorylation, is synthesized by virtually all cells, but how eukaryotes make the universal CoQ head group precursor 4-hydroxybenzoate (4-HB) from tyrosine is unknown. The first and last steps of this pathway have been defined in Saccharomyces cerevisiae, but the intermediates and enzymes involved in converting 4-hydroxyphenylpyruvate (4-HPP) to 4-hydroxybenzaldehyde (4-HBz) have not been described. Here, we interrogate this pathway with genetic screens, targeted LC-MS, and chemical genetics. We identify three redundant aminotransferases (Bna3, Bat2, and Aat2) that support CoQ biosynthesis in the absence of the established pathway tyrosine aminotransferases, Aro8 and Aro9. We use isotope labeling to identify bona fide tyrosine catabolites, including 4-hydroxyphenylacetate (4-HPA) and 4-hydroxyphenyllactate (4-HPL). Additionally, we find multiple compounds that rescue this pathway when exogenously supplemented, most notably 4-hydroxyphenylacetaldehyde (4-HPAA) and 4-hydroxymandelate (4-HMA). Finally, we show that the Ehrlich pathway decarboxylase Aro10 is dispensable for 4-HB production. These results define new features of 4-HB synthesis in yeast, demonstrate the redundant nature of this pathway, and provide a foundation for further study.

In Vitro Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe.

The gut bacterial genotoxin colibactin is linked to the development of colorectal cancer. In the final stages of colibactin's biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.