Production, homology modeling and mutagenesis studies on GlcH glucose transporter from Prochlorococcus sp. strain SS120.

Autor: Moreno-Cabezuelo JÁ; Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Córdoba, Spain., Del Carmen Muñoz-Marín M; Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Córdoba, Spain., López-Lozano A; Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Córdoba, Spain., Athayde D; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal., Simón-García A; Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Córdoba, Spain., Díez J; Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Córdoba, Spain., Archer M; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal., Issoglio FM; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal; CONICET-Universidad de Buenos Aires, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Buenos Aires, Argentina. Electronic address: fissoglio@qb.fcen.uba.ar., García-Fernández JM; Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Córdoba, Spain. Electronic address: jmgarcia@uco.es.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Bioenergetics [Biochim Biophys Acta Bioenerg] 2023 Apr 01; Vol. 1864 (2), pp. 148954. Date of Electronic Publication: 2022 Dec 20.
DOI: 10.1016/j.bbabio.2022.148954
Abstrakt: The marine cyanobacterium Prochlorococcus is one of the main primary producers on Earth, which can take up glucose by using the high affinity, multiphasic transporter GlcH. We report here the overexpression of glcH from Prochlorococcus marinus strain SS120 in Escherichia coli. Modeling studies of GlcH using the homologous MelB melibiose transporter from Salmonella enterica serovar Typhimurium showed high conservation at the overall fold. We observed that an important structural interaction, mediated by a strong hydrogen bond between D8 and R141, is conserved in Prochlorococcus, although the corresponding amino acids in MelB from Salmonella are different. Biased docking studies suggested that when glucose reaches the pocket of the transporter and interacts with D8 and R141, the hydrogen bond network in which these residues are involved could be disrupted, favoring a conformational change with the subsequent translocation of the glucose molecule towards the cytoplasmic region of the pmGlcH structure. Based on these theoretical predictions and on the conservation of N117 and W348 in other MelB structures, D8, N117, R141 and W348 were mutated to glycine residues. Their key role in glucose transport was evaluated by glucose uptake assays. N117G and W348G mutations led to 17 % decrease in glucose uptake, while D8G and R141G decreased the glucose transport by 66 % and 92 % respectively. Overall, our studies provide insights into the Prochlorococcus 3D-structure of GlcH, paving the way for further analysis to understand the features which are involved in the high affinity and multiphasic kinetics of this transporter.
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 © 2022. Published by Elsevier B.V.)
Databáze: MEDLINE
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