Identification and functional evaluation of Leishmania braziliensis Nicotinamide Mononucleotide Adenylyltransferase

• Published in: Protein expression and purification 2015-11, Vol.115, p.26-33
• Main Authors: Contreras, Luis E., Neme, Rafik, Ramírez, María H.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Escherichia coli - genetics; Leishmania; Leishmania braziliensis - enzymology; Leishmania braziliensis - genetics; Models, Molecular; Molecular Sequence Data; NAD - analysis; NAD - metabolism; Nicotinamide adenine dinucleotide (NAD+); Nicotinamide Mononucleotide Adenylyltransferase (NMNAT); Nicotinamide-Nucleotide Adenylyltransferase - chemistry; Nicotinamide-Nucleotide Adenylyltransferase - genetics; Nicotinamide-Nucleotide Adenylyltransferase - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.08.022

•The NMNAT enzymes of different Leishmania species were identified.•The enzymatic activity of L. braziliensis NMNAT was confirmed.•Leishmania NMNAT contains a specific amino-terminal insertion.•Deletion of the insertion is negatively correlated with in vitro enzymatic activity.•We suggest the amino-terminal insertion as a promising pharmacological target. The progressive increase in Leishmania resistance to current control approaches prompts the need to develop therapeutic strategies based on comprehensive knowledge of the parasite’s biology. The enzyme Nicotinamide Mononucleotide Adenylyltransferase (NMNAT, EC 2.7.7.1) catalyzes the central step in nicotinamide adenine dinucleotide (NAD+) biosynthesis, making it essential for the survival of all organisms. NAD+ metabolism is related to the maintenance of several biochemical, cellular, and physiological processes; consequently, the characterization and analysis of the enzymes involved in its biosynthesis represent key steps in the development of control strategies. In this study, the NMNAT enzymes of different Leishmania species were identified using bioinformatics procedures. The sequences were used to construct structural homology models that revealed characteristic elements common to NMNATs. The open reading frame of Leishmania braziliensis NMNAT was cloned from complementary DNA and the enzymatic activity of the corresponding recombinant protein was confirmed through enzymatic assays. Primary structure analysis revealed a Leishmania-specific amino-terminal insertion in NMNAT. The deletion of this insertion is negatively correlated with in vitro enzymatic activity. From our observations, we suggest the amino-terminal insertion of Leishmania NMNATs as a promising pharmacological target for the development of specific control strategies.