title: Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.
abstract: Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).
authors.affiliation: Department of Chemistry, University of Manchester Institute of Science and Technology (UMIST), Manchester, UK.
authors.forename: Gabriel C
authors.initials: GC
authors.lastname: Uguru
authors.forename: Claire
authors.initials: C
authors.lastname: Milne
authors.forename: Matthew
authors.initials: M
authors.lastname: Borg
authors.forename: Fiona
authors.initials: F
authors.lastname: Flett
authors.forename: Colin P
authors.initials: CP
authors.lastname: Smith
authors.forename: Jason
authors.initials: J
authors.lastname: Micklefield
mesh_terms: D000225:Adenine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D008401:Gas Chromatography-Mass Spectrometry; D007476:Ionophores / Q000737:chemistry; D015394:Molecular Structure; D010453:Peptide Synthases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D015202:Protein Engineering / Q000379:methods*; D012270:Ribosomes / Q000737:chemistry*; D013302:Streptomyces; D013869:Thiolester Hydrolases / Q000737:chemistry*
chemical_list: C040487:CDA antibiotic; D007476:Ionophores; D010455:Peptides; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D000225:Adenine