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

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.

Fig 1. NMN deamidase activity and NAD⁺ salvage pathway in A. tumefaciens.

A) Nicotinamide mononucleotide deamidase reaction. NMN is enzymatically transformed into nicotinic acid mononucleotide and ammonia. B) NAD⁺ salvage pathway in A. tumefaciens. Enzymes are indicated by the acronym used to identify the corresponding gene name. NadD, NaMN adenylyltransferase; NadE, NAD synthetase; NadM, NMN adenyltransferase; NadR^N, NMN adenylyltransferase; NadV, NAM phosphoribosyltransferase; PncA, nicotinamidase; PncB, nicotinic acid phosphoribosyltransferase; CinA, nicotinamide mononucleotide deamidase. The NCBI locus tag for each enzyme in A. tumefaciens is shown inside the corresponding square. NAD⁺, nicotinamide adenine dinucleotide; NAM, nicotinamide; NA: nicotinic acid; NaMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide; and NaAD, nicotinic acid adenine dinucleotide.

Fig 2. Multiple sequence alignment of A. tumefaciens nicotinamide mononucleotide deamidase with its homologues.

ESPript outputs of CinA after alignment by Clustal Omega with the corresponding sequences from Agrobacterium tumefaciens CinA (AtCinA, UniProt code: A9CJ26), Escherichia coli CinA (EcCinA, UniProt code: P0A6G3), Propionibacterium shermanii CinA (PsCinA, UniProt code: D7GE75), Azotobacter vinelandii CinA (AvCinA, UniProt code: C1DSQ5), Salmonella typhimurium CinA (StCinA, UniProt code: Q8ZMK4), Thermus thermophilus PncC (TtPncC, UniProt code: Q5SHB0), Oceanobacillus iheyensis PncC (OiPncC, UniProt code: Q8EQR8), Shewanella oneidensis PncC (SoPncC, UniProt code: Q8EK32), and Bacillus subtilis PncC (BsPncC, UniProt code: P46323). Residues strictly conserved across NMN deamidase enzymes are highlighted against a red background. The secondary structure corresponding to the crystallized A. tumefaciens NMN deamidase (2A9S) is shown, where springs represent helices and arrows represent β-strands. The most conserved residues in the CinA family are marked with triangles.

Fig 3. Circos representation of the NMN deamidase family (PF02464).

The outer ring shows the amino acid code corresponding to AtCinA (2A9S). Colored square boxes of the second circle indicate the KL (Kullback-Leibler) conservation score (from red to cyan, red: highest; cyan: lowest) [28]. The third and fourth circles show the cMI (cumulative Mutual Information score) and pMI (proximal Mutual Information score) scores as histograms, facing outward and inward, respectively. Lines in the center of the circle connect pairs of positions with MI (Mutational Information) score >6.5. Red lines represent the top 5%; black ones are between 70 and 95%, while grey ones account for the last 70% [28]. Names of the blocks correspond to those previously described [19], whereas the bold letters are the conserved amino acids.

Fig 4. Effect of pH and temperature on AtCinA activity.

A) pH profile for AtCinA. The assay conditions at 37°C were 0.5 mM NMN and 14 nM purified AtCinA. The buffers used (50 mM) were sodium phosphate (pH 6.0–7.5), Tris-HCl (pH 8.0) and glycine-NaOH (pH 9.0–10.0). Sodium acetate at pH 5.0 was used at 160 mM in order to maintain the same ionic strength. B) pH-stability. AtCinA was incubated at 37°C for different periods of time at different pHs, and the activity was measured under standard conditions. The buffers used (50 mM) were sodium phosphate pH 6.0 (■), pH 7.0 (▲), pH 7.5 (▼), Tris-HCl pH 8.0 (○), glycine pH 9.0 (∕) and pH 10.0 (△), except for sodium acetate pH 5.0 (●), which was at 160 mM as in A). C) Temperature profile for AtCinA activity. Conditions were the same as above, but at different temperatures (20–90°C) in 50 mM sodium phosphate pH 7.5. D) Thermal stability at pH 7.5. AtCinA was incubated for different periods of time at different temperatures [4°C (●), 25°C (■), 35°C (▲), 40°C (▼), 50°C (○), 60°C (∕), and 70°C (ρ)], and the activity was measured in the standard reaction conditions.

Fig 5. Thermal shift assay of AtCinA.

Melting temperature curves of purified enzyme (10 μg) were obtained in the presence of fluorescent probe SYPRO Orange in MilliQ^® water (◆), 160 mM sodium acetate pH 5.0 (●), 50 mM sodium phosphate pH 7.5 (▼), and 50 mM glycine pH 10.0 (■). Inset. Effect of different modulators on the melting temperature of AtCinA. Nucleotides were used at 1 mM (NAD⁺, FAD and NaMN), whereas hydroxy-ectoine (OH-Ect) and ammonium sulfate (AS) were used at 2 M and 1.5 M, respectively. Differences in ΔTm were calculated by subtracting the MilliQ^® water Tm value from the Tm values obtained for the enzyme in the different conditions used.

Fig 6. Effect of NMN concentration on AtCinA activity.

The activity was measured in the standard reaction conditions in the presence of increasing concentrations of up to 1 mM NMN, except in the case of Y58F mutant, when the concentration ranged from 0 to 10 mM (upper X-scale). Enzyme concentrations were 14 nM for wild type (●), 17 nM for C32 mutant (▼), 14 nM for S48A mutant (▲), 20 nM for T105A mutant (■), and 658 nM for Y58F mutant (◆).

Fig 7. NaMN binding site.

A) AtCinA binding site. NaMN (light cyan) was placed in the AtCinA structure (2A9S) using a structural alignment with the corresponding TtPncC structure (4UOC) [22] with Chimera [32]. B) TtPncC binding site with crystalized NaMN (cyan) (4UOC) [22]. Hydrogen bonds are shown in magenta. Distances are displayed as dashed lines.