engleski

ATPase activity of non-ribosomal peptide synthetases

Introduction: A variety of bioactive natural peptide products is produced by several bacterial and fungal species via the protein thiotemplate mechanism catalysed by non-ribosomal peptide synthetases (NRPS). NRPSs are composed of modular proteins, each module consisting of a set of apparently independent structural and functional domains that bind, activate and condense the carboxyl substrate into the growing polypeptide chain. Although a large body of information is available on the structure-function relationship of peptide synthetases, much of the basic aspects concerning the molecular dynamics in maintaining amino acid recognition specificity and regulation of the biosynthetic process still remain unclear. In this work, analysis of the anticipated ATPase activity of NRPS was examined. To assess whether this activity is relevant in the presence of the amino acid substrate, the reaction was followed in the presence of a number of naturally occurring amino acids and amino acid analogues known to support variable degrees of adenylation efficiency. Materials and Methods: Apo-tyrocidine synthetase 1 (apo-TY1), gramicidin S synthetase 2 (GS2) and δ -(L-α -aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), were purified from recombinant E. coli, Bacillus brevis ATCC9999 and Acremonium chrysogeneum, respectively. A coupled spectrophotometric assay involving purine nucleoside phosphorylase assay was applied to follow the continuous turnover of Pi in the presence of NRPS, ATP and amino acid. Results: Besides ATP-dependent adenylation activity, here we show that multifunctional NRPS demonstrate an ATPase activity, in the range of up to 2 Pi/min. Whereas the cognate amino acids display, in general, a negative effect on the ATPase activity, the presence of non-cognate substrates increases the rate of Pi generation. The effect is less pronounced with ACSV, which is a complete biosynthetic system, than with apo-TY1 (monomodular component of the tyrocidine biosynthetic system catalysing the activation of a single amino acid, L-Phe) or GS2 (tetramodular component catalysing the activation of L-Orn, L-Pro, L-Leu and L-Val). The enhanced hydrolysis rate by apo-TY1 in the presence of non-cognate amino acid substrates correlates well with their structural features and the diminishing adenylation efficiency. Conclusion: The adenylation domain of the multifunctional NRPSs demonstrates, besides ATP-dependent adenylation activity, an additional property related to ATP binding and hydrolysis, which might be part of the substrate recognition mechanism. This knowledge may prove useful in exploring the regulatory mechanism of peptide synthetases, relevant in exploiting their combinatorial potential for the generation of novel and more effective variants of existing drugs, or using the non-ribosomal chemistry for the preprogrammed generation of new peptide-forming systems.

adenylation domain; amino acid specificity; ATP binding; Pi release

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