Characterization of bacterial NMN deamidase as a Ser/Lys hydrolase expands diversity of serine amidohydrolases

Abstract

Edited by Miguel De la RosaKeywords: NMN deamidase Pyridine nucleotide Catalytic dyad Amidohydrolase Site-directed mutagenesis a b s t r a c t NMN deamidase (PncC) is a bacterial enzyme involved in NAD biosynthesis. We have previously demonstrated that PncC is structurally distinct from other known amidohydrolases. Here, we extended PncC characterization by mutating all potential catalytic residues and assessing their individual roles in catalysis through kinetic analyses. Inspection of these residues' spatial arrangement in the active site, allowed us to conclude that PncC is a serine-amidohydrolase, employing a Ser/Lys dyad for catalysis. Analysis of the PncC structure in complex with a modeled NMN substrate supported our conclusion, and enabled us to propose the catalytic mechanism.

NAD homeostasis in the bacterial response to DNA/RNA damage

“…The most common pathway recycling NMN released by the DNA ligase reaction is initiated by the enzyme NMN deamidase (PncC), which deamidates NMN to NaMN [61,62]. NaMN is then converted to NaAD by NaMN adenylyltransferase of the NadD family, and NaAD is finally amidated to NAD by NAD synthetase (NadE) (Fig.…”

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

“…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].…”

“…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.…”

“…Based on these latter studies and the mutational analysis carried out with E . coli CinA [23], two different catalytic mechanisms have been proposed [22,23], one describing a catalytic Ser/Lys dyad mechanism [23] and the other suggesting an asparaginase II-like mechanism [24]. In this last mechanism, a water nucleophilic attack is promoted by a lysine (K301 in TtPncC) on the amide bond, and the resulting tetrahedral intermediate is rearranged to eliminate ammonia with the assistance of a threonine (T340 in TtPncC) [22].…”

Mycobacterial Nicotinate Mononucleotide Adenylyltransferase

“…These enzymes also drive recycling of intracellular nicotinamide resulting from NAD utilization by CobB (and possibly other, yet unknown, N-glycosyl hydrolases). Recycling of NMN, another NAD degradation product generated by the NAD-dependent DNA ligase, is likely mediated by a recently characterized enzyme NMN deamidase, directly converting it to NaMN (39,40).…”