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.
Highlights
The enzyme nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42) catalyzes the conversion of NMN to nicotinic acid mononucleotide (NaMN) and ammonia (Figure 1, red line). Such activity was first described in Salmonella typhimurium and Escherichia coli in the late 70s, as one of the reactions implicated in the Pyridine Nucleotide Cycle (PNC), which in turn, is associated with bacterial NAD+ salvage (Figure 1) [1,2,3]
In most cases, such as in the case of the NMN deamidase from Agrobacterium tumefaciens (UniProt code: A9CJ26; PBD code: 2A9S) or Escherichia coli YgaD (UniProt code: P0A6G3), NMN deamidases are formed by just one domain containing the deamidase activity, the so-called PncC domain or competence/damage-inducible protein A (CinA) domain (Figure S1)
PncC domain is fused in its Nterminal with a MocF domain, which exhibits some degree of homology with enzymes involved in the last step of molybdenum cofactor (Moco) biosynthesis
Summary
The enzyme nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42) catalyzes the conversion of NMN to nicotinic acid mononucleotide (NaMN) and ammonia (Figure 1, red line). Such activity was first described in Salmonella typhimurium and Escherichia coli in the late 70s, as one of the reactions implicated in the Pyridine Nucleotide Cycle (PNC), which in turn, is associated with bacterial NAD+ salvage (Figure 1) [1,2,3]. Its product NaMN is the connection point between de novo routes and the most commonly occurring NAD+ salvage pathway, the Preiss-Handler pathway (Figure 1, shadowed reactions) This NMN deamidase has been related to the prevention of the inhibition of bacterial NAD+-.
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