Biocatalytic in Vitro and in Vivo FMN Prenylation and (De)carboxylase Activation

Allbwn ymchwil: Cyfraniad at gyfnodolynErthygladolygiad gan gymheiriaid

Fersiynau electronig

Dangosydd eitem ddigidol (DOI)

  • Khorcheska A Batyrova
    University of Toronto
  • Anna N Khusnutdinova
    Department of Chemical Engineering and Applied SciencesUniversity of TorontoInstitute of Basic Biological Problems of the Russian Academy of Sciences
  • Po-Hsiang Wang
    University of Toronto
  • Rosa Di Leo
    University of Toronto
  • Robert Flick
    University of Toronto
  • Elizabeth A Edwards
    University of Toronto
  • Alexei Savchenko
    University of Toronto
  • Alexander F Yakunin
    University of Toronto

Reversible UbiD-like (de)carboxylases represent a large family of mostly uncharacterized enzymes, which require the recently discovered prenylated FMN (prFMN) cofactor for activity. Functional characterization of novel UbiDs is hampered by a lack of robust protocols for prFMN generation and UbiD activation. Here, we report two systems for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions. The in vitro one-pot prFMN cascade includes five enzymes: FMN prenyltransferase (UbiX), prenol kinase, polyphosphate kinase, formate dehydrogenase, and FMN reductase, which use prenol, polyphosphate, formate, ATP, NAD+, and FMN as substrates and cofactors. Under aerobic conditions, this cascade produced prFMN from FMN with over 98% conversion and activated purified ferulic acid decarboxylase Fdc1 from Aspergillus niger and protocatechuic acid decarboxylase ENC0058 from Enterobacter cloaceae. The in vivo system for FMN prenylation and UbiD activation is based on the coexpression of Fdc1 and UbiX in Escherichia coli cells under aerobic conditions in the presence of prenol. The in vitro and in vivo FMN prenylation cascades will facilitate functional characterization of novel UbiDs and their applications.

Allweddeiriau

Iaith wreiddiolSaesneg
Tudalennau (o-i)1874-1882
Nifer y tudalennau9
CyfnodolynACS Chemical Biology
Cyfrol15
Rhif y cyfnodolyn7
Dyddiad ar-lein cynnar24 Meh 2020
Dynodwyr Gwrthrych Digidol (DOIs)
StatwsCyhoeddwyd - 17 Gorff 2020
Cyhoeddwyd yn allanolIe
Gweld graff cysylltiadau