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

Ana Belén Martínez-Moñino,Fernando Gil-Ortiz,Manuela Pérez-Gilabert,Álvaro Sánchez-Ferrer,Rubén Zapata-Pérez,Antonio Ginés García-Saura,Shihui Yang

doi:10.1371/journal.pone.0174759

Ana Belén Martínez-Moñino, Fernando Gil-Ortiz + Show 5 more

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https://doi.org/10.1371/journal.pone.0174759

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Abstract

NAD+ has emerged as a crucial element in both bioenergetic and signaling pathways since it acts as a key regulator of cellular and organismal homeostasis. Among the enzymes involved in its recycling, nicotinamide mononucleotide (NMN) deamidase is one of the key players in the bacterial pyridine nucleotide cycle, where it catalyzes the conversion of NMN into nicotinic acid mononucleotide (NaMN), which is later converted to NAD+ in the Preiss-Handler pathway. The biochemical characteristics of bacterial NMN deamidases have been poorly studied, although they have been investigated in some firmicutes, gamma-proteobacteria and actinobacteria. In this study, we present the first characterization of an NMN deamidase from an alphaproteobacterium, Agrobacterium tumefaciens (AtCinA). The enzyme was active over a broad pH range, with an optimum at pH 7.5. Moreover, the enzyme was quite stable at neutral pH, maintaining 55% of its activity after 14 days. Surprisingly, AtCinA showed the highest optimal (80°C) and melting (85°C) temperatures described for an NMN deamidase. The above described characteristics, together with its high catalytic efficiency, make AtCinA a promising biocatalyst for the production of pure NaMN. In addition, six mutants (C32A, S48A, Y58F, Y58A, T105A and R145A) were designed to study their involvement in substrate binding, and two (S31A and K63A) to determine their contribution to the catalysis. However, only four mutants (C32A, S48A Y58F and T105A) showed activity, although with reduced catalytic efficiency. These results, combined with a thermal and structural analysis, reinforce the Ser/Lys catalytic dyad mechanism as the most plausible among those proposed.

Highlights

During the last decade, it has become clear that the cellular role of NAD+ extends far beyond its classic participation in redox reactions, since it acts as a substrate of several families of regulatory enzymes [1,2,3,4]
A. tumefaciens nicotinamide mononucleotide (NMN) deamidase-encoding protein (AtCinA) was found in the UniProt database (A9CJ26) as a 169 amino acids putative competence/damage inducible 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
The NMN deamidase from Agrobacterium tumefaciens was cloned in E. coli, overexpressed, and purified to obtain a stable 41-kDa homodimeric protein

Summary

Introduction

It has become clear that the cellular role of NAD+ extends far beyond its classic participation in redox reactions, since it acts as a substrate of several families of regulatory enzymes [1,2,3,4] For this reason, intracellular concentrations of many NAD+ intermediates are maintained low [5,6,7,8], and NAD+ is considered as a sensor of metabolic and cellular stress [9,10,11]. Its gene (pncC) was sequenced in the marine bacterium Shewanella oneidensis MR-1 [18], and matched with a family of genes annotated in databases as competence/damageinducible protein A (cinA). The function of CinA protein in this process is still a riddle, it could play a dual role in decreasing the intracellular level of NMN to avoid inhibition of NAD+-dependent DNA ligases, and at the same time, securing a continuous NAD+ supply for the ligases by propelling NaMN into the Preiss-Handler pathway to produce new NAD+ [18]

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