title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 MURCIA, Spain.|Murcia Biomedical Research Institute (IMIB), Murcia, Spain.
authors.forename: Ana Belén
authors.initials: AB
authors.lastname: Martínez-Moñino
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 MURCIA, Spain.|Murcia Biomedical Research Institute (IMIB), Murcia, Spain.
authors.forename: Rubén
authors.initials: R
authors.lastname: Zapata-Pérez
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 MURCIA, Spain.|Murcia Biomedical Research Institute (IMIB), Murcia, Spain.
authors.forename: Antonio Ginés
authors.initials: AG
authors.lastname: García-Saura
authors.affiliation: CELLS-ALBA Synchrotron Light Source, Barcelona, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Gil-Ortiz
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 MURCIA, Spain.|Murcia Biomedical Research Institute (IMIB), Murcia, Spain.
authors.forename: Manuela
authors.initials: M
authors.lastname: Pérez-Gilabert
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 MURCIA, Spain.|Murcia Biomedical Research Institute (IMIB), Murcia, Spain.
authors.forename: Álvaro
authors.identifier: http://orcid.org/0000-0001-7266-4402
authors.initials: Á
authors.lastname: Sánchez-Ferrer
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D000581:Amidohydrolases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D017386:Sequence Homology, Amino Acid
chemical_list: D000581:Amidohydrolases; C021861:nicotinamidenucleotide amidase