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Analysis of the primary structure of peptide synthetases involved in
non-ribosomal synthesis of peptide antibiotics revealed a highly conserved and
ordered domain structure. These functional units, which are about 1000 amino
acids in length, are believed to be essential for amino acid activation and
thioester formation. To delineate the minimal extension of such a domain, we
have amplified and cloned truncated fragments of the grsA gene, encoding the
1098-amino acid multifunctional gramicidin S synthetase 1, GrsA. The
overexpressed His6-tagged GrsA derivatives were affinity-purified, and the
catalytic properties of the deletion mutants were examined by biochemical
studies including ATP-dependent amino acid activation, carboxyl thioester
formation, and the ability to racemize the covalently bound phenylalanine from
L- to the D-isomer. These studies revealed a core fragment (PheAT-His) that
comprises the first 656 amino acid residues of GrsA, which restored all
activities of the native protein, except racemization of phenylalanine. A
further deletion of about 100 amino acids at the C-terminal end of the GrsA core
fragment (PheAT-His), including the putative thioester binding motif LGGHSL,
produced a 556-amino acid fragment (PheA-His) that shows a
phenylalanine-dependent aminoacyl adenylation, but almost no thioester
formation. A 291-amino acid deletion at the C terminus of the native GrsA, that
contains a putative racemization site resulted in complete loss of racemization
ability (PheATS-His). However, it retained the functions of specific amino acid
activation and thioester formation. The results presented defined biochemically
the minimum size of a peptide synthetase domain and revealed the locations of
the functional modules involved in substrate recognition and ATP-dependent
activation as well as in thioester formation and racemization.
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