Editing of non-cognate aminoacyl adenylates by peptide synthetases

Abstract

Non-ribosomally formed peptides display both highly conserved and variable amino acid positions, the variations leading to a wide range of peptide families. Activation of the amino acid substrate proceeds in analogy to the ribosomal biosynthetic mechanism generating aminoacyl adenylate and acyl intermediates. To approach the mechanism of fidelity of amino acid selection, the stability of the aminoacyl adenylates was studied by employing a continuous coupled spectrophotometric assay. The apo-form of tyrocidine synthetase 1 (apo-TY1) was used, generating an -phenylalanyl-adenylate intermediate stabilized by the interaction of two structural subdomains of the adenylation domain. Adenylates of substrate analogues have shown variable and reduced degrees of stability, thus leading to an enhanced generation of pyrophosphate due to hydrolysis and continuous adenylate formation. Discrimination of the non-

Dipeptide formation on engineered hybrid peptide synthetases

“…that A domains are autonomous units that retain their catalytic potential even when overproduced as distinct proteins [19]. Our results, however, provide evidence that the exact activation pattern of an A domain is a function of an active-site configuration involving adjacent domain interaction [29]. The importance of conformational changes in the process of adenylation and substrate release has been discussed recently [24].…”

“…The importance of conformational changes in multidomain modules during the process of amino-acid adenylation and product release has been discussed previously [29]. It is possible that the introduction of fusion sites could impair these conformational changes.…”

“…The formation of thioesters is a crucial step in the investigation of the hybrid NRPSs used in this study, because this step directly involves substrate transfer between domains of different origin. Only a proper interaction between the A and PCP domains ensures significant loading of substrate amino acids [29]. Previous heterologous loading experiments in trans with dissected A and PCP domains were obviously hampered by poor domain-domain interactions, which revealed an impaired substrate thiolation [30].…”

The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides

“…This relaxed substrate specificity occurs more commonly in the recognition and activation of hydrophobic amino acids [35]. A‐domains, however, do appear to have some editing ability to prevent the activation of non‐substrate amino acids by the hydrolysis of the intermediate aminoacyl adenylate [37].…”