RESUMO
The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a 'Trojan horse' antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligases/química , Ligases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Antibacterianos/biossíntese , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Bacteriocinas/biossíntese , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Humanos , Ligases/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Nucleotídeos/química , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
Synthetic phosphoramidate analogues of nucleosides have been used as enzyme inhibitors for decades and have therapeutic applications in the treatments of HIV and cancer, but little is known about how N-P bonds are fashioned in nature. The heptapeptide MccA undergoes post-translational processing in producer strains of Escherichia coli to afford microcin C7 (MccC7), a "Trojan horse" antibiotic that contains a phosphoramidate linkage to adenosine monophosphate at its C-terminus. We show that the enzyme MccB, encoded by the MccC7 gene cluster, is responsible for formation of the N-P bond in MccC7. This modification requires the consumption of two ATP molecules per MccA peptide and formation and breakdown of a peptidyl-succinimide intermediate.