Heterologous overexpression and characterization of homoserine dehydrogenase from Paracoccidioides brasiliensis.

Autor: Santos JL; São Carlos Institute of Chemistry, Universidade de São Paulo, São Carlos, SP, Brazil., Ângelo EA; Instituto Federal do Paraná, Umuarama, PR, Brazil., Gauze GF; Department of Chemistry, Universidade Estadual de Maringá, Maringá, PR, Brazil., Seixas FAV; Department of Technology, Universidade Estadual de Maringá, Umuarama, PR, Brazil., Canduri F; São Carlos Institute of Chemistry, Universidade de São Paulo, São Carlos, SP, Brazil. Electronic address: fcanduri@iqsc.usp.br.
Jazyk: angličtina
Zdroj: Biochimie [Biochimie] 2023 Aug; Vol. 211, pp. 87-95. Date of Electronic Publication: 2023 Mar 17.
DOI: 10.1016/j.biochi.2023.03.003
Abstrakt: 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 α-helices and 10.5 ± 7.0% β-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectroscopy has shown stability of PbHSD in the presence of urea until C m 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 T m  = 58.65 ± 0.87 °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 K M of 224 ± 15 μM and specific activity at optimum pH of 2.10 ± 0.07 μmol/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.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)
Databáze: MEDLINE
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