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Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli
BACKGROUND: Biotin is an essential enzyme cofactor that acts as a CO(2 )carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2010
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964542/ https://www.ncbi.nlm.nih.gov/pubmed/20937134 http://dx.doi.org/10.1186/1472-6750-10-73 |
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author | Delli-Bovi, Teegan A Spalding, Maroya D Prigge, Sean T |
author_facet | Delli-Bovi, Teegan A Spalding, Maroya D Prigge, Sean T |
author_sort | Delli-Bovi, Teegan A |
collection | PubMed |
description | BACKGROUND: Biotin is an essential enzyme cofactor that acts as a CO(2 )carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for (35)S labeling of biotin. RESULTS: In this study, we produced [(35)S]-biotin from Na(35)SO(4 )and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. CONCLUSIONS: The strategy described in our report provides a simple and effective method for the production of [(35)S]-biotin in E. coli based on affinity chromatography. |
format | Text |
id | pubmed-2964542 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-29645422010-10-28 Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli Delli-Bovi, Teegan A Spalding, Maroya D Prigge, Sean T BMC Biotechnol Research Article BACKGROUND: Biotin is an essential enzyme cofactor that acts as a CO(2 )carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for (35)S labeling of biotin. RESULTS: In this study, we produced [(35)S]-biotin from Na(35)SO(4 )and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. CONCLUSIONS: The strategy described in our report provides a simple and effective method for the production of [(35)S]-biotin in E. coli based on affinity chromatography. BioMed Central 2010-10-11 /pmc/articles/PMC2964542/ /pubmed/20937134 http://dx.doi.org/10.1186/1472-6750-10-73 Text en Copyright ©2010 Delli-Bovi et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Delli-Bovi, Teegan A Spalding, Maroya D Prigge, Sean T Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli |
title | Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli |
title_full | Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli |
title_fullStr | Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli |
title_full_unstemmed | Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli |
title_short | Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli |
title_sort | overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in e. coli |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964542/ https://www.ncbi.nlm.nih.gov/pubmed/20937134 http://dx.doi.org/10.1186/1472-6750-10-73 |
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