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

Abstract

Nicotinamide mononucleotide (NMN) deamidase is one of the key enzymes of the bacterial pyridine nucleotide cycle (PNC). It catalyzes the conversion of NMN to nicotinic acid mononucleotide, which is later converted to NAD+ by entering the Preiss-Handler pathway. However, very few biochemical data are available regarding this enzyme. This paper represents the first complete molecular characterization of a novel NMN deamidase from the halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiPncC). The enzyme was active over a broad pH range, with an optimum at pH 7.4, whilst maintaining 90 % activity at pH 10.0. Surprisingly, the enzyme was quite stable at such basic pH, maintaining 61 % activity after 21 days. As regard temperature, it had an optimum at 65 °C but its stability was better below 50 °C. OiPncC was a Michaelian enzyme towards its only substrate NMN, with a K m value of 0.18 mM and a kcat/K m of 2.1 mM-1 s-1. To further our understanding of these enzymes, a complete phylogenetic and structural analysis was carried out taking into account the two Pfam domains usually associated with them (MocF and CinA). This analysis sheds light on the evolution of NMN deamidases, and enables the classification of NMN deamidases into 12 different subgroups, pointing to a novel domain architecture never before described. Using a Logo representation, conserved blocks were determined, providing new insights on the crucial residues involved in the binding and catalysis of both CinA and MocF domains. The analysis of these conserved blocks within new protein sequences could permit the more efficient data curation of incoming NMN deamidases.

Characterization and mutational analysis of a nicotinamide mononucleotide deamidase from Agrobacterium tumefaciens showing high thermal stability and catalytic efficiency

“…tumefaciens NMN deamidase-encoding protein (AtCinA) was found in the UniProt database (A9CJ26) as a 169 amino acids putative c ompetence/damage in ducible protein A (CinA protein). When it was compared with other NMN deamidases described in the bibliography ( Fig 2 ), AtCinA showed moderate degree of amino acid sequence identity with proteins from Salmonella typhimurium (UniProt entry: Q8ZMK4) [33], Azotobacter vinelandii (UniProt entry: C1DSQ5) [20], Escherichia coli (UniProt entry: P0A6G3) [23], Shewanella oneidensis (UniProt entry: Q8EK32) [18], Propionibacterium shermanii (UniProt entry: D7GE75) [21], Bacillus subtilis (UniProt entry: P46323) [34], Thermus thermophilus (UniProt entry: Q5SHB0) [22] and Oceanobacillus iheyensis (UniProt entry: Q8EQR8) [19], with a 53%, 49%, 47%, 41%, 38%, 37%, 35% and 30% sequence identity, respectively.…”

“…These positions, also conserved in Fig 2 (green triangles), indicate that AtCinA has all the described amino acids that form part of the active site [18,19,22,23], including those belonging to the described block P-I (E30, S31, C32, T33, G35) [19] ( Fig 3 ; S1 Fig , red), of which S31 and C32 have been related with the catalysis and stabilization of the tetrahedral catalytic intermediate, respectively [23]. The backbone amides of G46 and S48 belonging to the described block P-II [19] are involved in hydrogen bonding with O3P and O2P atoms of the substrate/product (NMN/NaMN) phosphate group, whereas Y58 (block P-III) interacts with O1P ( S1 Fig ) [22]. K63 (block P-III) ( Fig 3 ; S1 Fig ) has been described as acting as a general base in both proposed NMN deamidase catalytic mechanisms [22,23].…”

“…In fact, only five have been kinetically characterized [13,18–21] and their nomenclature is confusing, since two of the terms “PncCs” and “CinAs” are used indistinctively, while they are in fact different. Phylogenetic and domain analyses have shown that the term PncC is better applied to enzymes formed by a domain with NMN deamidase activity (CinA domain) fused to another domain with ADP-ribose (ADPr) pyrophosphatase or NUDIX ( Nu cleoside Di phosphate linked to X ) hydrolase activity (MocF domain), whereas the term CinA is better applied to NMN deamidases with only one domain [18,19]. …”

“…In addition, a MocF domain structure has also been resolved from Thermoplasma acidophilum (PDB code: 3KBQ). ADPr and NMN substrates were docked to the above structures [18,19], and NaMN and ADPr were co-crystallized [22] in order to identity the amino acids involved in binding and catalysis. Based on these latter studies and the mutational analysis carried out with E .…”

“…This enzyme was selected because it belongs to a biochemically uncharacterized subgroup (2.2.3) in the general phylogenetic tree described for the NMN deamidase family [19]. In addition, no NMN deamidases from an alphaproteobacteria have been previously characterized.…”