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Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba

Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a...

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Autores principales: Xia, Jikun, Xin, Wanmeng, Wang, Fang, Xie, Wancui, Liu, Yi, Xu, Jiakun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9499490/
https://www.ncbi.nlm.nih.gov/pubmed/36142390
http://dx.doi.org/10.3390/ijms231810478
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author Xia, Jikun
Xin, Wanmeng
Wang, Fang
Xie, Wancui
Liu, Yi
Xu, Jiakun
author_facet Xia, Jikun
Xin, Wanmeng
Wang, Fang
Xie, Wancui
Liu, Yi
Xu, Jiakun
author_sort Xia, Jikun
collection PubMed
description Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli (E. coli). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei. Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA’s enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA.
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spelling pubmed-94994902022-09-23 Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba Xia, Jikun Xin, Wanmeng Wang, Fang Xie, Wancui Liu, Yi Xu, Jiakun Int J Mol Sci Article Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli (E. coli). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei. Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA’s enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA. MDPI 2022-09-09 /pmc/articles/PMC9499490/ /pubmed/36142390 http://dx.doi.org/10.3390/ijms231810478 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Xia, Jikun
Xin, Wanmeng
Wang, Fang
Xie, Wancui
Liu, Yi
Xu, Jiakun
Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba
title Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba
title_full Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba
title_fullStr Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba
title_full_unstemmed Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba
title_short Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba
title_sort cloning and characterization of fructose-1,6-bisphosphate aldolase from euphausia superba
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9499490/
https://www.ncbi.nlm.nih.gov/pubmed/36142390
http://dx.doi.org/10.3390/ijms231810478
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