Heterologous overexpression and characterization of homoserine dehydrogenase from Paracoccidioides brasiliensis

被引:2
作者
Santos, Jessyka Lima [1 ]
Angelo, Elisangela Andrade [2 ]
Gauze, Gisele de Freitas [3 ]
Seixas, Flavio Augusto Vicente [4 ]
Canduri, Fernanda [1 ,5 ]
机构
[1] Univ Sao Paulo, Sao Carlos Inst Chem, Sao Carlos, SP, Brazil
[2] Inst Fed Parana, Umuarama, PR, Brazil
[3] Univ Estadual Maringa, Dept Chem, Maringa, PR, Brazil
[4] Univ Estadual Maringa, Dept Technol, Umuarama, PR, Brazil
[5] Univ Sao Paulo, Inst Quim Sao Carlos, Ave Trab Sao Carlense,400 Parque Arnold Schimidt, BR-13566590 Sao Carlos, SP, Brazil
关键词
Paracoccidioidomycosis; Homoserine dehydrogenase; Specific activity; CIRCULAR-DICHROISM SPECTRA; PROTEIN-STRUCTURE;
D O I
10.1016/j.biochi.2023.03.003
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:87 / 95
页数:9
相关论文
共 21 条
[1]   New inhibitors of homoserine dehydrogenase from Paracoccidioides brasiliensis presenting antifungal activity [J].
Alves Bueno, Paulo Sergio ;
Vilugron Rodrigues, Franciele Abigail ;
Santos, Jessyka Lima ;
Canduri, Fernanda ;
Biavatti, Debora Carina ;
Pimentel, Arethusa Lobo ;
Bagatin, Mariane Cristovao ;
Kioshima, Erika Seki ;
Gauze, Gisele de Freitas ;
Vicente Seixas, Flavio Augusto .
JOURNAL OF MOLECULAR MODELING, 2019, 25 (11)
[2]  
Bagatin MC, 2017, ANTIMICROB AGENTS CH, V61, DOI [10.1128/AAC.00165-17, 10.1128/aac.00165-17]
[3]   QUANTITATIVE-ANALYSIS OF PROTEIN FAR UV CIRCULAR-DICHROISM SPECTRA BY NEURAL NETWORKS [J].
BOHM, G ;
MUHR, R ;
JAENICKE, R .
PROTEIN ENGINEERING, 1992, 5 (03) :191-195
[4]  
Cordova L. A., 2021, Paracoccidioidomycosis. StatPearls
[5]  
da Silva L., 2005, CARACTERIZACAO ESTUD, DOI [10.17771/PUCRio.acad.8015, DOI 10.17771/PUCRIO.ACAD.8015]
[6]  
Gasteiger E., 2005, PROTEIN ANAL TOOLS E, P571, DOI [https://doi.org/10.1385/1-59259-890-0:571, DOI 10.1385/1-59259-890-0:571]
[7]   Using circular dichroism spectra to estimate protein secondary structure [J].
Greenfield, Norma J. .
NATURE PROTOCOLS, 2006, 1 (06) :2876-2890
[8]   The Use of Circular Dichroism in the Investigation of Protein Structure and Function [J].
Kelly, Sharon M. ;
Price, Nicholas C. .
CURRENT PROTEIN & PEPTIDE SCIENCE, 2000, 1 (04) :349-384
[9]  
Ladokhin A.S., 2000, ENCY ANAL CHEM APPL, P1, DOI [10.1002/9780470027318.a1611, DOI 10.1002/9780470027318.A1611]
[10]   New Trends in Paracoccidioidomycosis Epidemiology [J].
Martinez, Roberto .
JOURNAL OF FUNGI, 2017, 3 (01)