دورية أكاديمية
أعرض في EDS

An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal target from Candida albicans.

التفاصيل البيبلوغرافية
العنوان: An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal target from Candida albicans.
المؤلفون: 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.; Genesilico Biotech, Brasília, DF, Brazil., de Freitas SM; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil., Felipe MSS; Programa de Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, 70790-160, Brazil., Barbosa JARG; Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil. joaobarbosa@unb.br.
المصدر: Fungal biology and biotechnology [Fungal Biol Biotechnol] 2024 May 07; Vol. 11 (1), pp. 5. Date of Electronic Publication: 2024 May 07.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: BioMed Central Country of Publication: England NLM ID: 101655873 Publication Model: Electronic Cited Medium: Internet ISSN: 2054-3085 (Electronic) Linking ISSN: 20543085 NLM ISO Abbreviation: Fungal Biol Biotechnol Subsets: PubMed not MEDLINE
أسماء مطبوعة: Original Publication: London : BioMed Central, [2014]-
مستخلص: 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 β-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.
(© 2024. The Author(s).)
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معلومات مُعتمدة: 0193.001533/2016 Fundação de Apoio à Pesquisa do Distrito Federal - FAP/DF; 0193.001533/2016 Fundação de Apoio à Pesquisa do Distrito Federal - FAP/DF; 0193.001533/2016 Fundação de Apoio à Pesquisa do Distrito Federal - FAP/DF; 422508/2021-7 Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq; 422508/2021-7 Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq; 422508/2021-7 Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq
فهرسة مساهمة: Keywords: Candida albicans; Biofilm; Drug resistance; Drug target; Extracellular matrix; Glycosylation; Mnt1/Kre2; Structural properties; α-1,2-mannosyltransferase
تواريخ الأحداث: Date Created: 20240507 Latest Revision: 20240510
رمز التحديث: 20240510
مُعرف محوري في PubMed: PMC11077754
DOI: 10.1186/s40694-024-00174-5
PMID: 38715132
قاعدة البيانات: MEDLINE
الوصف
تدمد:2054-3085
DOI:10.1186/s40694-024-00174-5