Production and molecular characterization of NAD+ related enzymes and their advanced intermediates

Abstract

In the PhD Thesis entitled "Production and molecular characterization of NAD+-related enzymes and their advanced intermediates", the objectives were: the development of an enzymatic synthesis method in order to obtain pure nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR); the design of a functional screening method to discover new nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries; the application of the developed functional screening to find new extremophile metagenomic nicotinamidases/pyrazinamidases; the analysis of the black truffle genome to assign a putative protein as a nicotinamidase/pyrazinamidase based on its functional characterization; and the biochemical and structural characterization of a hypothetical protein from Oceanobacillus iheyensis as a MacroD-like macrodomain. To obtain pure NMN and NR, a bacterial NAD+-diphosphatase and 5'-nucleotidase were cloned and purified. The first enzyme was able to convert NAD+ into NMN and AMP, being the latter compound separated from NMN by ion exchange chromatography. The NMN obtained was fully transformed into NR by the 5'-nucleotidase. Furthermore, both compounds were tested and compared with commercial NMN in two cellular models, finding not only the same NAD+ increase in one of the cell types but also, for the first time, a higher increment in the NAD+ levels of the other cellular model treated with our enzymatic compounds. For the functional screening of nicotinamidases/pyrazinamidases, two new whole-cell methods were developed using the chemical property of the products formed in the enzymatic reaction catalyzed by nicotinamidases (pyrazinoic or nicotinic acids) to form colored complexes with the stable iron salts ammonium ferrous sulfate (AFS) or sodium nitroprusside (SNP). After the optimization of the conditions, a fosmid polygenomic library was screened, discovering several positive clones with the AFS method. Their quantitative re-screening with the SNP method allowed us to discover the first nicotinamidase with balanced catalytic efficiency towards nicotinamide and pyrazinamide. Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of nicotinamidase inhibitors by the naked eye. Using the previously developed method, together with bioinformatics, the first hyperthermophilic nicotinamidase from an unclassified bacterium (UbNic) was found, with an optimum temperature of 90 °C and a broad optimum pH. The enzyme showed one of the highest catalytic efficiencies among non-pathogenic bacterial nicotinamidases. Furthermore, the sequence of the metal binding site revealed that UbNic and its related sequences belong to a subgroup not described so far. With the knowledge acquired in the field of nicotinamidases, a bioinformatic analysis was performed to identify a new gene from truffle as a putative nicotinamidase. Its presence in the mycelium of the fungus was demonstrated with the AFS method developed before. After its recombinant expression and purification, a biochemical characterization of the protein was carried out, finding that this nicotinamidase has a clear preference for its natural substrate (NAM) than for pyrazinamide. The enzyme also had a unique inhibition pattern with different aldehydes. Finally, the functional and structural characterization of the macrodomain from Oceanobacillus iheyensis allowed us to shed light on its substrate binding and catalysis. The crystal structures of D40A, N30A and G37V mutants, and those with MES, ADP-ribose and ADP bound, led us to the identification of five fixed water molecules that play a significant role in substrate-binding. Furthermore, the closure of the ?6-?4 loop was revealed as essential for pyrophosphate recognition and distal ribose orientation. In addition, a novel structural role for residue D40 was identified. Moreover, it was revealed that OiMacroD catalyzes the hydrolysis of O-acetyl-ADP-ribose and also reverses protein mono-ADP-ribosylation. Finally, mutant G37V supports the participation of a substrate-coordinated water molecule in catalysis that helps to select the proper substrate conformation.