RESUMO
Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.
Assuntos
Aminoaciltransferases , Peptidil Transferases , Aminopeptidases , Proteínas de Bactérias/metabolismo , Aminoaciltransferases/metabolismo , Peptídeos/metabolismoRESUMO
Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.
Assuntos
Aminoaciltransferases , Depsipeptídeos , Staphylococcus aureus , Aminoaciltransferases/química , Proteínas de Bactérias/química , Glicina , Indicadores e ReagentesRESUMO
The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of ß-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked ß-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.
Assuntos
Acetilcoenzima A/metabolismo , Carboxiliases/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Glutamato Descarboxilase/metabolismo , Modelos Moleculares , Acetilcoenzima A/análogos & derivados , Acetilcoenzima A/química , Substituição de Aminoácidos , Antibacterianos/farmacologia , Antimetabólitos/farmacologia , Sítios de Ligação , Calorimetria , Carboxiliases/química , Carboxiliases/genética , Coenzima A/síntese química , Coenzima A/química , Coenzima A/metabolismo , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Deleção de Genes , Glutamato Descarboxilase/antagonistas & inibidores , Glutamato Descarboxilase/química , Glutamato Descarboxilase/genética , Cinética , Mutação , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/farmacologia , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TitulometriaRESUMO
Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.