The Escherichia coli NadR regulator is endowed with nicotinamide mononucleotide adenylyltransferase activity

Abstract

The first identification and characterization of a catalytic activity associated with NadR protein is reported. A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases. The Escherichia coli nadR gene was cloned into a T7-based vector and overexpressed. In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.

FIG. 1

Sequence alignment of homologous regions of NadR proteins from E. coli (ENadR) and S. typhimurium (SNadR), NMN adenylyltransferase from M. jannaschii (MjAT), putative NMN adenylyltransferases from Methanobacterium thermoautotrophicum (MtAT), Archaeoglobus fulgidus (AfAT), and Pyrococcus horikoshii (PhAT), and NMN adenylyltransferase from Synechocystis sp. (SynAT). Identical and similar residues are in boldface. The alignment was generated by using the CLUSTAL W program.

FIG. 2

Purification of recombinant NadR protein. A Coomassie blue-stained polyacrylamide gel (10%) shows the fractions from the purification: (3 μg of hydroxyapatite fraction [lane a], 5 μg of DNA agarose fraction [lane b], and 28 μg of crude extract [lane c]), and positions of molecular weight standards (2 μg; lane d).

FIG. 3

Gel retardation of the nadB operator region by NadR protein. An ethidium bromide-stained polyacrylamide gel shows the migration of a 200-bp DNA fragment containing the nadB control region (60 nM) after incubation with increasing amounts of pure NadR. Concentrations of NadR were 211 nM (lane a), 114 nM (lane b), 55 nM (lane c), and 0 nM (lane d).