Characterization of new molecules and enzymes involved in the regulation of NAD+ levels

Abstract

In the PhD Thesis entitled "Characterization of new molecules and enzymes involved in the regulation of NAD+ levels", the objectives were: the design of a new method for the classification of the great number of nicotinamidases present in databases, in order to find new singular nicotinamidases; the development of a biotechnological method for the production of NAD+-boosters using new bacterial NAD+ diphosphatases; the in silico study and characterization of new non-bacterial NAD+ diphosphatases; the characterization of a new bacterial poly-ADP-ribose polymerase with high activity; and the characterization of a new bacterial macrodomain enzyme able to remove poly-ADP-ribose polymers. In order to classify the members of the nicotinamidase family, a Prosite-compatible pattern was designed. This pattern based on the metal binding site, common for all the nicotinamidases, allowed to identify more than ten thousand sequences. Based on the above described pattern, four new sub-patterns were developed, including those of Firmicutes-like, Mycobacteria-like, Archaea-like and Multi-origin-like pattern. This new classification of nicotinamidases based on their metal binding site also correlates well with that obtained when the complete sequences is used, pointing out for the first time a method for the classification of nicotinamidases. For the NAD+-boosters production a new bacterial NAD+ diphosphatase was purified and immobilized in the form of BSA-CLEAs. The important increase in operational stability and reusability of these BSA-CLEAs make possible its use for NAD+-booster compounds production (NMN). In addition, when this biocatalyst was combined with an NMN deamidase from E. coli, a simple process for obtaining NaMN was obtained. This enzymatically synthetized NaMN was tested in two different cell lines, hepatocytes and adipocytes, giving raise a clear increase in the intracellular NAD+ content, demonstrating for the first time that NaMN can be used as a NAD+-booster compound. Following the previous interest in improving the NAD+-booster compounds production, a new fungal NAD+ diphosphatase was biochemically characterized after a deep bioinformatics analysis. This alkaliphic enzyme, showed a high catalytic efficiency, which was several fold higher than those described in the bibliography including those of bacteria, and with the exception of that of human (hNUDT12). However, its NAD+/NADH catalytic efficiency ratio was higher than hNUDT12, giving rise to the first example of an enzyme with this profile, and pointing out the biotechnological potential of this new enzyme. The search for a new bacterial poly-ADP-ribose polymerase led to the discovery of the enzyme from Clostridioides difficile CD160. This enzyme was found to be a highly active enzyme, 3-fold more than that of the bacteria Herpetosiphon auranticus. In addition, the first phylogenetic analysis of bacterial PARPs was also performed, showing that CdPARP has a singular domain organization compared with those found in other clostridial PARPs, which could be due to a long-term divergence of C. difficile CD160 and related to its unique toxigenotype (A-B+CDT-). C. difficile CD160 has become of great interest due to the fact that this microorganism has also a functional bona fide PAR metabolism, not only with both active poly-ADP-ribose polymerase and glycohydrolase but also with putative MacroD protein. The major discovery in this study was a new PARP inhibitor, never before used with human PARP, that selectively inhibited hPARP1 but not the bacterial PARPs, indicating a high degree of selectivity. Finally, a new bacterial macrodomain from Fusobacterium mortiferum ATCC 9817 was described, being the first bacterial macrodomain belonging to the OARD1-type macrodomains, which are able to carry out three different enzymatic reactions, deacetylation of OAADPr, de-MARylation and de-PARylation. The ability of this new macrodomain for removing the complete poly(ADP-ribose) polymer (PAR) from PARylated proteins is of great importance, since this activity has only been substantiated in human OARD1 macrodomain.