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Pdx2 employs a Cys\u2010His\u2010Glu catalytic triad to deaminate glutamine to glutamate and ammonia \u2013 the source of the nitrogen of pyridoxal 5\u2019\u2010phosphate (PLP). This enzyme is, therefore, a rate\u2010limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens that rely on this mechanism to obtain PLP. For this reason, Pdx2 is considered a novel and promising drug target to treat these diseases. The results obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i)\u2005activation of Cys<jats:sub>87<\/jats:sub>, (ii)\u2005deamination of glutamine with the formation of the glutamyl\u2010thioester intermediate, (iii)\u2005hydrolysis of the formed intermediate, and (iv)\u2005enzymatic turnover. The kinetic data available in the literature (19.1\u201319.5\u2005kcal\u2009mol<jats:sup>\u22121<\/jats:sup>) agree very well with the calculated free energy barrier of the hydrolytic step (18.2\u2005kcal.mol<jats:sup>\u221211<\/jats:sup>), which is the rate\u2010limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i)\u2005it requires the activation of the nucleophile Cys<jats:sub>87<\/jats:sub> to a thiolate; ii)\u2005the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii) Glu<jats:sub>198<\/jats:sub> does not have a direct role in the catalytic process. Together, these results can be used for the synthesis of new transition state analogue inhibitors capable of inhibiting Pdx2 and impair diseases like Malaria and Tuberculosis.<\/jats:p>","DOI":"10.1002\/cbic.202100555","type":"journal-article","created":{"date-parts":[[2021,11,11]],"date-time":"2021-11-11T13:10:41Z","timestamp":1636636241000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys\u2010His\u2010Glu Triad: A Computational Study"],"prefix":"10.1002","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9773-0808","authenticated-orcid":false,"given":"Andr\u00e9 F.","family":"Pina","sequence":"first","affiliation":[{"name":"Associate Laboratory i4HB \u2013 Institute for Health and Bioeconomy Faculty of Medicine University of Porto  4200-319 Porto Portugal"},{"name":"UCIBIO \u2013 Applied Molecular Biosciences Unit BioSIM \u2013 Department of Biomedicine Faculty of Medicine University of Porto  4200-319 Porto Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6560-5284","authenticated-orcid":false,"given":"S\u00e9rgio F.","family":"Sousa","sequence":"additional","affiliation":[{"name":"Associate Laboratory i4HB \u2013 Institute for Health and Bioeconomy Faculty of Medicine University of Porto  4200-319 Porto Portugal"},{"name":"UCIBIO \u2013 Applied Molecular Biosciences Unit BioSIM \u2013 Department of Biomedicine Faculty of Medicine University of Porto  4200-319 Porto Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0342-7424","authenticated-orcid":false,"given":"Nuno M. 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