Journal of the American Chemical Society · 2004 · 61 citations · 11 references
EngineeringNonribosomal Peptide SynthetaseMolecular BiologyDonor SiteActive-site ModificationsChemical BiologyEnzymatic ModificationProtein SynthesisBiosynthesisStructure-function Enzyme KineticsSite-directed MutagenesisBiochemistryAdenylation Domains LeadProtein BiosynthesisNatural SciencesPeptide LibraryPeptoidEnzyme CatalysisSynthetic BiologyPeptide SynthesisProtein Engineering
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).
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Functional Arteries Grown in Vitro
Laura E. Niklason, Jinming Gao, William M. Abbott et al. · Science · 1999 · 1.7K citations