AI Article Synopsis
- The enzyme Homoserine dehydrogenase (PbHSD) from Paracoccidioides brasiliensis was successfully expressed in E. coli, yielding 30 mg per liter of culture with high purity.
- Structural analysis indicates that PbHSD consists of about 45.5% α-helices and 10.5% β-strands, and it functions as a homodimer.
- Kinetic studies show that PbHSD has optimal activity at pH 9.35-9.50, with a Michaelis constant (K) of 224 μM, demonstrating its potential for development as an antifungal drug.
Article Abstract
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 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 = 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 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.
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| http://dx.doi.org/10.1016/j.biochi.2023.03.003 | DOI Listing |
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