1H, 15N, 13C backbone resonance assignment of Escherichia coli AlkB

Abstract

Escherichia coli enzyme AlkB has been extensively studied as the prototypical enzyme of the AlkB family of α-ketoglutarate dependent dioxygenases that reverse nucleotide and protein alkylation resulting from mutagen exposure and cell-signaling. Upon binding of the secondary co-substrates Fe(II) and α-ketoglutarate, AlkB transitions from a conformationally dynamic, to a well defined tertiary structure that has been crystallized extensively. Upon binding of the alkylated substrate, a nucleotide recognition lid stabilizes the complex, promoting the hydroxylation of the alkyl lesion and reversal of the substrate to its unmodified state. However, the mechanism by which this takes place is not well understood. Here we report the 1H, 15N, 13C backbone assignment of the 24kDa AlkB from E. coli. Assignments were obtained at 21 °C by heteronuclear multidimensional NMR spectroscopy. In total 82% of all backbone resonances were assigned with 161 out of 197 assigned in the 1H-15N TROSY spectrum. The secondary structure prediction generated from the assigned backbone resonances using TALOS+ is in good agreement with existing crystal structures of AlkB. The reported assignment will allow further structural and thermodynamic investigation of the regulatory mechanisms at play in ligand binding, and how they are coupled to conformational dynamics in AlkB.