Genomics-guided analysis of NAD recycling yields functional elucidation of COG1058 as a new family of pyrophosphatases

PLoS One. 2013 Jun 12;8(6):e65595. doi: 10.1371/journal.pone.0065595. Print 2013.

Abstract

We have recently identified the enzyme NMN deamidase (PncC), which plays a key role in the regeneration of NAD in bacteria by recycling back to the coenzyme the pyridine by-products of its non redox consumption. In several bacterial species, PncC is fused to a COG1058 domain of unknown function, highly conserved and widely distributed in all living organisms. Here, we demonstrate that the PncC-fused domain is endowed with a novel Co(+2)- and K(+)-dependent ADP-ribose pyrophosphatase activity, and discuss the functional connection of such an activity with NAD recycling. An in-depth phylogenetic analysis of the COG1058 domain evidenced that in most bacterial species it is fused to PncC, while in α- and some δ-proteobacteria, as well as in archaea and fungi, it occurs as a stand-alone protein. Notably, in mammals and plants it is fused to FAD synthase. We extended the enzymatic characterization to a representative bacterial single-domain protein, which resulted to be a more versatile ADP-ribose pyrophosphatase, active also towards diadenosine 5'-diphosphate and FAD. Multiple sequence alignment analysis, and superposition of the available three-dimensional structure of an archaeal COG1058 member with the structure of the enzyme MoeA of the molybdenum cofactor biosynthesis, allowed identification of residues likely involved in catalysis. Their role has been confirmed by site-directed mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Diphosphate Ribose / metabolism*
  • Amidohydrolases / genetics
  • Amidohydrolases / metabolism*
  • Base Sequence
  • Cloning, Molecular
  • Computational Biology
  • Genomics / methods
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NAD / metabolism*
  • Phylogeny
  • Protein Structure, Tertiary / physiology
  • Pyrophosphatases / genetics
  • Pyrophosphatases / metabolism*
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Species Specificity

Substances

  • NAD
  • Adenosine Diphosphate Ribose
  • Amidohydrolases
  • nicotinamidenucleotide amidase
  • Pyrophosphatases

Grants and funding

This research was partly supported by the Italian Ministry of Foreign Affairs, “Direzione Generale per la Promozione del Sistema Paese” to NR; by the U.S. Department of Energy (DOE), Office of Biological and Environmental Research (BER) Genomic Science Program (GSP) as part of the Pacific Northwest National Laboratory (PNNL) Foundational Scientific Focus Area to ALO; and by The Ministry of Education and Science of Russian Federation, projects 8135 and 8049 to MDK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.