Directed Evolution of the BpsA Carrier Protein Domain for Recognition by Non-cognate 4'-Phosphopantetheinyl Transferases to Enable Inhibitor Screening

Methods Mol Biol. 2023:2670:145-163. doi: 10.1007/978-1-0716-3214-7_7.

Abstract

4'-Phosphopantetheinyl transferases (PPTases) play an essential role in activating the carrier protein domains of mega-synthases involved in primary and secondary metabolism and have been validated as promising drug targets in multiple pathogens. Monitoring phosphopantetheinylation of the non-ribosomal peptidase synthetase BpsA, which produces blue indigoidine pigment upon activation, is a useful strategy to screen chemical collections for inhibitors of a target PPTase. However, PPTases can exhibit carrier protein specificity and some medically important PPTases do not activate BpsA. Here, we describe how to conduct a directed evolution campaign to evolve the BpsA carrier protein domain for improved recognition by a candidate PPTase, as exemplified for the human Sfp-like PPTase. This method can be applied to other non-cognate PPTases for discovery of new drug candidates or chemical probes, or to enable development of next-generation biosensors that utilize BpsA as a reporter.

Keywords: BpsA; Directed evolution; Error-prone PCR; Human PPTase; Indigoidine; PPTase; Peptidyl carrier-protein domain; Targeted screening.

MeSH terms

  • Bacterial Proteins / metabolism
  • Carrier Proteins* / metabolism
  • Humans
  • Transferases (Other Substituted Phosphate Groups) / metabolism
  • Transferases* / metabolism

Substances

  • Carrier Proteins
  • Transferases
  • Transferases (Other Substituted Phosphate Groups)
  • Bacterial Proteins