UC San Diego

Phosphopantetheine (PPant) transfer from coenzyme A (CoA) to carrier protein (CP) is a necessary post-translational modification required for much natural product biosynthesis. Since this enzymatic modification is essential to many pathogens, high-throughput detection of phosphopantetheinyl transferase (PPTase) activity in a fluorescence polarization format enables methodology to identify new antimicrobial compounds. The ability of the PPTase to utilize fluorescent analogs of phosphopantetheine and the CoA from which it is derived is critical to activity detection. However, the utility of appending phosphopantetheine labels extends past the application of fluorophores to a CP. Many other types of labels can be appended to a phosphopantetheine and attached to CP and short peptide substrates, such as affinity groups, substrate mimics, and crosslinking agents, all enabling various novel biochemical applications. Until recently, the facile removal of natural and synthetic PPant from CP was not possible. However, with the characterization of a stable acyl carrier protein hydrolase (AcpH) phosphodiesterase from the bacteria Pseudomonas aeruginosa, reversible labeling of the acyl carrier protein (ACP) was enabled, allowing new bioconjugation methods to label and regenerate both native and fusion CP. Further work with new AcpH homologs from the bacteria Pseudomonas fluorescens and cyanobacteria Cyanothece sp. PCC 7822 reveal enzymes with superior kinetic parameters and CP substrate promiscuity extending to small peptide activity