Abstract
Nudix (for nucleoside diphosphatases linked to other moieties, X) hydrolases are a diverse family of proteins capable of cleaving an enormous variety of substrates, ranging from nucleotide sugars to NAD+-capped RNAs. Although all the members of this superfamily share a common conserved catalytic motif, the Nudix box, their substrate specificity lies in specific sequence traits, which give rise to different subfamilies. Among them, NADH pyrophosphatases or diphosphatases (NADDs) are poorly studied and nothing is known about their distribution. To address this, we designed a Prosite-compatible pattern to identify new NADDs sequences. In silico scanning of the UniProtKB database showed that 3% of Nudix proteins were NADDs and displayed 21 different domain architectures, the canonical architecture (NUDIX-like_zf-NADH-PPase_NUDIX) being the most abundant (53%). Interestingly, NADD fungal sequences were prominent among eukaryotes, and were distributed over several Classes, including Pezizomycetes. Unexpectedly, in this last fungal Class, NADDs were found to be present from the most common recent ancestor to Tuberaceae, following a molecular phylogeny distribution similar to that previously described using two thousand single concatenated genes. Finally, when truffle-forming ectomycorrhizal Tuber melanosporum NADD was biochemically characterized, it showed the highest NAD+/NADH catalytic efficiency ratio ever described.
Highlights
Nudix hydrolases are a diverse family of proteins capable of cleaving an enormous variety of substrates, ranging from nucleotide sugars to NAD+-capped RNAs
We have carried out an extensive bioinformatic analysis, taking into account other important amino acids recently described in the EcNudC structure6,23, giving rise to a new Prosite-compatible NADH pyrophosphatases or diphosphatases (NADDs) pattern, which expands the previously published NADH diphosphatase sequence array
Based on the relevance of the above mentioned amino acids, the catalytic residues in the Nudix box (E174, E177 and E178) (Fig. 1, squares) and the essential role of NudC dimerization in substrate recognition, the Prosite-compatible pattern [CS]x(2)[CD] x(12,15)[CN]x(5,35)[YF]Px[3]Px[2]Ix[25,32]GFx[4]Ex[7]REx[2]EEx[13,14]Q[PQ]W[PA]xPx[2,9][QLIMA] M was designed (Fig. 1, red letters; Supplementary Fig. S1C). This pattern covers the distance between C98 and M201, and basically represents the zinc domain ([CS]x(2)[CD]x(12,15)[CN]x(5,35)[YF]) and the conserved amino acids in the Nudix domain without β16 and α3 (Fig. 1), which includes the Nudix Box (GFx[4]Ex[7] REx[2]EE) and part of the conserved eight-amino acid motif, known as NADD signature (Q[PQ]W[PA]xP) (Supplementary Fig. S1D)
Summary
Nudix (for nucleoside diphosphatases linked to other moieties, X) hydrolases are a diverse family of proteins capable of cleaving an enormous variety of substrates, ranging from nucleotide sugars to NAD+-capped RNAs. Nudix or NUDT (Nudix-type) hydrolases are Mg2+/Mn2+-dependent enzymes active towards nucleoside diphosphates linked to other moieties (X) and forming a superfamily distributed throughout the phylogenetic scale with more than 200,000 entries in the UniProtKB database They share the characteristic conserved sequence required for substrate catalysis named the Nudix box (GX5EX7REUXEEXGU), where U represents a bulky hydrophobic amino acid, usually Ile, Leu or Val. We have carried out an extensive bioinformatic analysis, taking into account other important amino acids recently described in the EcNudC structure, giving rise to a new Prosite-compatible NADD pattern, which expands the previously published NADH diphosphatase sequence array This in silico study provides the first phylogenetic distribution of NADDs, and gives a picture of their domain architectures. The cloning and kinetic characterization of T. melanosporum NADD uncovers a new efficient biocatalyst with the highest NAD+/NADH catalytic efficiency ratio ever described
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