Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.
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).
Uguru
Gabriel C
GC
Department of Chemistry, University of Manchester Institute of Science and Technology (UMIST), Manchester, UK.
Milne
Claire
C
Borg
Matthew
M
Flett
Fiona
F
Smith
Colin P
CP
Micklefield
Jason
J
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*
C040487:CDA antibiotic; D007476:Ionophores; D010455:Peptides; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D000225:Adenine