Furthermore, a 2.3 fold increase in transmembrane spanning proteins was achieved in comparisons to control non-enriched tissue from the same brain region, further confirming the method of enrichment. Nicotinamidase is an important metabolic enzyme that can hydrolyze nicotinamide to nicotinic acid, always involved in the nicotinamide adenine dinucleotide salvage pathway of several pathogenic microorganisms such as Borrelia burgdorferi, Brucella abortus, and Mycobacterium tuberculosis. Because of lacking a de novo NAD+ biosynthetic pathway like mammals, many pathogens have to rely on the transformation from host’s NAM to their own NAD+ by nicotinamidase to keep the stabilization of NAD+ concentrations. Consequently, the nicotinamidase activity is of particular importance for the viability of these pathogens. Recently, targeting NAD+ biosynthesis as an antibiotic approach has been the intense subject of scientific investigation. Furthermore, the more important reason why nicotinamidase has attracted GDC-0449 widespread attention is that the Mycobacterium tuberculosis nicotinamidase can convert the NAM analogue prodrug pyrazinamide into the bacteriostatic compound pyrazinoic acid, hence the alternative name, pyrazinamidase. PZA, in combination with rifampicin and isoniazid, is the first-line drug recommended by the World Health Organization for the treatment of tuberculosis. The addition of PZA to this regimen leads to a significant reduction in the length of chemotherapy, allowing the conventional 9-month tuberculosis treatment to be shortened to 6 months. The latest research has proved that the active form of PZA, POA, targets the ribosomal proteins S1 of M. tuberculosis, a vital protein in protein translation and the ribosome-sparing process of transtranslation, hence a subsequent inhibition of trans-translation and the shortened duration of tuberculosis chemotherapy. Despite the significance of PncA in the NAD+ salvage pathway and the PZA activation, its mechanism of action has not yet been fully understood. Since mutations in pncA gene were found by Scorpio and Zhang, a lot of researches have identified various mutations in pncA gene that can lead to the loss of PncA activity, which are thought to be highly correlated with the PZA resistance in M. tuberculosis. One of the most striking features of these mutations is their diversity, with hundreds of mutations scattered throughout the pncA gene, not only in the active site. In order to interpret how the mutations result in the PZA resistance, the structure/function relationships of PncA need to be deeply comprehended. In 2001, Du and colleagues established the unliganded crystal structure of PncA from Pyrococcus horikoshii, which shows that PhPncA has a Zn2+ ion coordinated by Asp52, His54 and His71, as well as a catalytic triad consist of Asp10, Lys94 and Cys133.