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Abstract
BackgroundBiotin 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.ResultsIn 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.ConclusionsThe strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.
Highlights
Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions
We found that coexpression of the E. coli biotin synthase, BioB, increased PfBCCP-79 biotinylation over basal levels, coexpression of both BioB and the E. coli biotin ligase, BirA, was required for efficient biotinylation of PfBCCP-79
Biotin is conjugated to a particular lysine in the biotin carboxy carrier protein (BCCP) domain
Summary
Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. Vitamin H, was first identified as a yeast growth factor over 100 years ago [1] and was subsequently isolated from egg yolk [2] and liver [3] It is an essential cofactor for a small family of enzymes that catalyze carboxylation and decarboxylation reactions, in which biotin serves as a covalent attachment site for CO2 [4]. Biotin-dependent enzymes include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase [6] These enzymes participate in ACC is the only biotinylated enzyme in Escherichia coli [9], and it exists as a complex of four proteins: biotin carboxylase (BC), carboxyl transferase alpha and beta chains (CT), and biotin carboxy carrier protein (BCCP) [10]. Biotin protein ligases show significant cross reactivity between species, i.e. bacterial ligases biotinylate mammalian apo-proteins, and vice versa [4]
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