Heterologous overexpression and characterization of homoserine dehydrogenase from Paracoccidioides brasiliensis

The enzyme Homoserine dehydrogenase from Paracoccidioides brasiliensis (PbHSD), an interesting enzyme in the search for new antifungal drugs against paracoccidioidomycosis, was expressed by E. coli. Thirty milligrams of PbHSD with 94% of purity were obtained per liter of culture medium. The analysis by CD spectroscopy indicates a composition of 45.5 +/- 7.3% of a-helices and 10.5 +/- 7.0% b-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectros-copy has shown stability of PbHSD in the presence of urea until Cm of 4.13 +/- 0.21 M, and a broad pH range in which there is no conformational change. The protein analysis by differential scanning calorimetry indicates high stability at room temperature, but low stability at high temperatures, suffering irreversible denaturation, with Tm = 58.65 +/- 0.87 degrees C. Kinetic studies of PbHSD by molecular absorption spectroscopy in UV/Vis have shown an optimum pH between 9.35 and 9.50, with Michaelian behavior, presenting KM of 224 +/- 15 mM and specific activity at optimum pH of 2.10 +/- 0.07 mmol/min/mg for homoserine. Therefore, protein expression and purification were efficient, and the structural characterization has shown that PbHSD presents native conformation with enzymatic activity in kinetic assays.(c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.