Forskning ved Københavns Universitet - Københavns Universitet


Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

  • Annabelle Hoegl
  • Matthew B Nodwell
  • Volker C Kirsch
  • Nina C Bach
  • Martin Pfanzelt
  • Matthias Stahl
  • Sabine Schneider
  • Stephan A Sieber

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2'-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.

TidsskriftNature Chemistry
Udgave nummer12
Sider (fra-til)1234-1245
Antal sider12
StatusUdgivet - dec. 2018
Eksternt udgivetJa

ID: 209575672