Reference - Detail
|Author||Delli-Bovi TA, Spalding MD, Prigge ST.|
|Title||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 CO2 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 35S labeling of biotin.
RESULTS:In this study, we produced [35S]-biotin from Na35SO4 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 [35S]-biotin in E. coli based on affinity chromatography.
|MeSH||Acetyl-CoA Carboxylase Biotin / analogs & derivatives Biotin / biosynthesis* Biotinylation Carbon-Nitrogen Ligases / genetics Carbon-Nitrogen Ligases / metabolism* Chromatography, Affinity Escherichia coli / enzymology* Escherichia coli / genetics Escherichia coli Proteins / genetics Escherichia coli Proteins / metabolism* Gene Expression Regulation, Bacterial Peptide Fragments Plasmodium falciparum / enzymology Repressor Proteins / genetics Repressor Proteins / metabolism* Sulfurtransferases / genetics Sulfurtransferases / metabolism*|
|WOS Category||BIOTECHNOLOGY & APPLIED MICROBIOLOGY|
|Prokaryotes E. coli||JW0758-KC|