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An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal target from Candida albicans.
Silva, Patrícia Alves; Souza, Amanda Araújo; de Oliveira, Gideane Mendes; Ramada, Marcelo Henrique Soller; Hernández, Nahúm Valente; Mora-Montes, Héctor Manuel; Bueno, Renata Vieira; Martins-de-Sa, Diogo; de Freitas, Sonia Maria; Felipe, Maria Sueli Soares; Barbosa, João Alexandre Ribeiro Gonçalves.
Afiliação
  • Silva PA; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil.
  • Souza AA; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil.
  • de Oliveira GM; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil.
  • Ramada MHS; Programa de Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, 70790-160, Brazil.
  • Hernández NV; Departmento de Biologia, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, 36050, Mexico.
  • Mora-Montes HM; Departmento de Biologia, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, 36050, Mexico.
  • Bueno RV; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil.
  • Martins-de-Sa D; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil.
  • de Freitas SM; Genesilico Biotech, Brasília, DF, Brazil.
  • Felipe MSS; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil.
  • Barbosa JARG; Programa de Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, 70790-160, Brazil.
Fungal Biol Biotechnol ; 11(1): 5, 2024 May 07.
Article em En | MEDLINE | ID: mdl-38715132
ABSTRACT

BACKGROUND:

Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking.

RESULTS:

We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and ß-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports.

CONCLUSIONS:

The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Fungal Biol Biotechnol Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Brasil País de publicação: Reino Unido

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Fungal Biol Biotechnol Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Brasil País de publicação: Reino Unido