H-1, N-15 and C-13 backbone resonance assignments of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2

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Abstract

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete 1H, 15N and 13C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the 1H–15N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism.

Original languageEnglish
Pages (from-to)79-83
Number of pages5
JournalBiomolecular NMR Assignments
Volume12
Issue number1
Early online date22 Nov 2017
DOIs
Publication statusPublished - Apr 2018

Keywords

  • Backbone resonance assignment
  • Flavin mononucleotide
  • Flavoenzyme
  • Pentaerythritol tetranitrate reductase
  • Transverse relaxation optimized spectroscopy

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology
  • Manchester Institute for Collaborative Research on Ageing

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