Pdx2, the glutaminase subunit of the pyridoxal 5’-phosphate (PLP) synthase, is a key enzyme in the synthesis of PLP. It employs a non-canonical Cys-His-Glu triad to catalyze the deamination of glutamine to glutamate and ammonia – the source of the nitrogen of PLP. For this reason, Pdx2 is considered a novel and promising drug target against diseases such as Malaria and Tuberculosis, whose pathogens rely on this enzyme to obtain PLP. Therefore, the catalytic mechanism of Pdx2 was studied with atomic detail using the computational ONIOM QM/MM methodology with an 80/81 atoms QM region, which includes all catalytic relevant residues, treated at the DLPNO-CCSD(T)/CBS//B3LYP/6-31G(d,p):ff14SB level. The results demonstrate that the catalytic mechanism of Pdx2 occurs in six steps divided into four main stages: (i) activation of Cys₈₇, (ii) deamination of glutamine with formation of the glutamyl-thioester intermediate, (iii) hydrolysis of the formed intermediate, and (iv) enzymatic turnover. The rate-limiting step of the complete catalytic mechanism is the hydrolysis of the glutamyl-thioester intermediate (18.2 kcal.mol^-1), which closely agrees with the available kinetic data (19.1–19.5 kcal mol^-1). The catalytic mechanism of Pdx2 differs from other known amidases in three main points: i) it requires the activation of the nucleophile Cys₈₇ to a thiolate; ii) the hydrolysis occurs in a single step without formation of a second tetrahedral intermediate, and iii) Glu₁₉₈ does not have a direct role in the catalytic process.