Genomics-Guided Analysis of NAD Recycling Yields Functional Elucidation of COG1058 as a New Family of Pyrophosphatases

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.

NAD homeostasis in the bacterial response to DNA/RNA damage

“…Therefore, a careful control of its cellular level is required, and such control can be accomplished by the activity of the ADPRP enzyme that hydrolyzes ADPR to AMP and R5P. Two evolutionary distinct ADPRPs seem to have been recruited by bacteria, one ADPRP belonging to the family of Nudix hydrolases [71], and a recently discovered ADPRP, classified as COG1058 in the Clusters of Orthologous Group Database [72]. Despite possessing a completely different fold and a distinct cofactor requirement, both ADPRPs display a comparable catalytic efficiency.…”

“…Moreover, both of them exhibit a peculiar tendency to occur in fused form with enzymes of the PNC, suggesting a functional connection between ADPR catabolism and NAD regeneration. In particular, in several bacterial species the Nudix ADPRP domain is fused to NadM [73,74] while in others the COG1058 ADPRP domain can be found fused to PncC [72]. It is conceivable that the enzymatic activities of bifunctional ADPRP/NadM and ADPRP/PncC would enable bacterial cells to coordinately recycle back to NAD both the pyridine and the ADPR moieties.…”

New Insights into the Phylogeny and Molecular Classification of Nicotinamide Mononucleotide Deamidases

“…However, structural and biochemical analysis showed these enzymes to be very different from each other: 2A9S contains the conserved residues responsible for NMN deamidase activity [7], whereas 3KBQ has been recently described as a new class of pyrophosphatase (eggNOG code: COG1058) [11]. When the structure of A. tumefaciens CinA (2A9S) was studied in detail using ArchSchema [12], a tool for interactive graphing of related Pfam domain architectures, three well-defined groups could be identified attending to domain composition (Figure 2).…”

Metabolism Dealing with Thermal Degradation of NAD + in the Hyperthermophilic Archaeon Thermococcus kodakarensis

“…tumefaciens ADPR-PPase belongs to clusters of orthologous groups (COG) that differ from that of TK2284 (30). As NMN deamidase is also known to function in NAD ϩ regeneration, this finding further supports that ADPR-PPase functions in a salvage pathway to supply NAD ϩ .…”