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.

Comparative inhibitory profile and distribution of bacterial PARPs, using Clostridioides difficile CD160 PARP as a model

“…The MISTIC server 27 (mutual information server to infer coevolution) was used to analyse and visualize the extent of the coevolutionary relationship between two positions in the bacterial PARP protein family by using the information contained within the above MSA (multiple sequence alignment) 28 . The mutual information (MI) obtained is usually used to find structurally or functionally important positions in a given protein fold family 29 , 30 . The residue-based Kullback-Leibler conservation score (Fig.…”

“…The thermal stability of the above PARPs was also studied under different conditions by monitoring Sypro Orange fluorescence while heating the samples, and determining the midpoints of the transitions (melting temperature or T m) 30 . Previous investigations showed that this assay provides a good estimate of binding affinity, optimal storage pH and the suitable protein stabilizers to be used 40 , 41 .…”

“…Protein melting curves to determine the thermal stability of PARPs were obtained using the fluorescent dye SYPRO Orange (Molecular Probes), as previous described 30 , 41 . Proteins (10 µg) were preincubated with 10X Sypro Orange (excitation at 490 nm, emission at 530 nm) in the presence of different buffers (100 mM), compounds (NAD + , ADP-ribose, nicotinamide, ammonium sulphate and hydroxyectoine) or inhibitors (3-aminobenzamidine).…”

An uncharacterized FMAG_01619 protein from Fusobacterium mortiferum ATCC 9817 demonstrates that some bacterial macrodomains can also act as poly-ADP-ribosylhydrolases

“…Fusobacterium mortiferum ATCC 9817 TARG1 macrodomain (Uniprot code, C3WDV1) was cloned into the pSol-AFV vector (Lucigen, USA), using the synthetic FMAG_01619 gene obtained from Genscript (USA), and transformed into Escherichia coli 10G 57 . The clone containing the FmTARG1 was grown in 1 L of Terrific Broth (TB) medium supplemented with kanamycin (50 µg mL −1 ).…”

Characterization of Resveratrol, Oxyresveratrol, Piceatannol and Roflumilast as Modulators of Phosphodiesterase Activity. Study of Yeast Lifespan

“…Although yPDE2 has previously been purified and characterized [7,9], this is the first time that the heterologous expression of this enzyme has been considered. E. coli was selected because it is easy to manage for protein expression [30]. The available UniProt sequence was used to construct an IPTG-induced vector and Rosetta 2 E. coli strain was transformed.…”