Chemistry & Biology
Volume 6, Issue 8, August 1999, Pages 493-505
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The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases

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Background

Many pharmacologically important peptides are synthesized nonribosomally by multimodular peptide synthetases (NRPSs). These enzyme templates consist of iterated modules that, in their number and organization, determine the primary structure of the corresponding peptide products. At the core of each module is an adenylation domain that recognizes the cognate substrate and activates it as its aminoacyl adenylate. Recently, the crystal structure of the phenylalanine-activating adenylation domain PheA was solved with phenylalanine and AMP, illustrating the structural basis for substrate recognition.

Results

By comparing the residues that line the phenylalanine-binding pocket in PheA with the corresponding moieties in other adenylation domains, general rules for deducing substrate specificity were developed. We tested these in silico ‘rules’ by mutating specificity-conferring residues within PheA. The substrate specificity of most mutants was altered or relaxed. Generalization of the selectivity determinants also allowed the targeted specificity switch of an aspartate-activating adenylation domain, the crystal structure of which has not yet been solved, by introducing a single mutation.

Conclusions

In silico studies and structure-function mutagenesis have defined general rules for the structural basis of substrate recognition in adenylation domains of NRPSs. These rules can be used to rationally alter the specificity of adenylation domains and to predict from the primary sequence the specificity of biochemically uncharacterized adenylation domains. Such efforts could enhance the structural diversity of peptide antibiotics such as penicillins, cyclosporins and vancomycins by allowing synthesis of ‘unnatural’ natural products.

Key words

adenylation domain
binding pocket
nonribosomal peptide synthetase
signature sequence
substrate specificity

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*

Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

1

Biochemie/Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Straβe, D-35032 Marburg, Germany.