Structure, function, and dynamics of vitamin B6 biosynthesis enzymes from Staphylococcus aureus

• Ano de defesa: 2023
• Autor(a) principal: Barra, Angélica Luana Carrillo
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Staphylococcus aureus; Bacterial resistance; Estado oligomérico; Oligomeric state; Resistência bacteriana; Síntese de vitamina B6; Vitamin B6 biosynthesis
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/76/76133/tde-11122023-092239/

The de novo synthesis of vitamin B6 (pyridoxal 5-phosphate) is conserved in most organisms but mammals. The synthesis of pyridoxal 5-phosphate (PLP) is carried out by a complex of two enzymes: Pdx1 and Pdx2. Pdx2 has glutaminase activity and transfers an ammonia molecule to Pdx1, which then utilizes ammonia, ribose 5-phosphate (R5P), and glyceraldehyde 3-phosphate (G3P) to synthesize PLP. There is no data of this pathway in Staphylococcus aureus, an opportunist pathogen of extreme concern. Hence, we propose investigating biochemical and structurally the bacterial Staphylococcus aureus PLP (SaPLP) synthase complex (SaPdx1- SaPdx2 complex) to bring light to its application as a potential target for antibiotics development. Therefore, the enzymes were expressed in Escherichia coli BL21 CodonPlus (DE3) RIL and purified through Ni-affinity chromatography, followed by TEV (Tobacco Etch Virus protease) cleavage and size exclusion chromatography (SEC). The oligomeric state of SaPdx1 in solution was analyzed using SEC-SAXS under three different conditions. The results revealed that the oligomerization of Pdx1 is dependent on salt concentration, reaching an equilibrium between dodecamers and hexamers under specific conditions. To better comprehend the biological significance of this phenomenon, SEC-MALS measurements were performed before and after the enzyme reaction. These experiments clarified that Pdx1 needs to assemble into a dodecamer to synthesize PLP. The crystallographic structure of SaPdx1 provided further insights, showing an ethylene glycol molecule bound to the active site, mimicking the substrate R5P interactions. Two monomers were found in asymmetric unit (ASU), but macromolecular interface analysis indicated that the dodecamer was the most probable quaternary structure. Regarding the SaPLP synthase complex, a mutation was introduced into SaPdx2, since it was described in literature that this mutation is related with the oligomerization of the PLP synthase complex. When examining the stability of the SaPLP synthase complex (wild type and mutant) through SEC-MALS and crystallographic structures, it was observed that the interaction in the wild type complex is transient and only fully saturated during catalysis. In contrast, the mutant complex exhibited greater stability, as expected. Kinetic assays revealed that the SaPLP synthase complex is more efficient than SaPdx1 when using alternative ammonia sources, evidencing the importance of SaPdx2 to the catalysis. For the first time, an inhibitory effect of high concentrations of G3P was observed, impacting SaPdx1 more than the complex. The three-dimensional structure of SaPdx1-2mut supports the hypothesis that the PLP synthase complex is fully occupied by glutaminase subunits through Pdx2 inactivation. Collectively, our data offer new insights to understand this complex pathway and provide valuable information for exploration in the field of drug discovery.