title: The A9 core sequence from NRPS adenylation domain is relevant for thioester formation.
abstract: The adenylation (A) domain in nonribosomal peptide synthetases catalyses a two-step reaction in which an amino acid is activated and then transferred to the neighbouring thiolation (T) domain. In this study, we investigated the role of the conserved A9 core sequence of the A-domain of tyrocidine synthetase 1, by analysis of single amino acid mutations in the A9 region. Mutation of an absolutely conserved proline (P490G) significantly reduced the conformational stability of the protein, as evidenced by increased susceptibility to proteolytic cleavage and denaturation. All mutant A-domains were capable of amino acid activation, but the activity in the overall reaction was reduced. Surprisingly, the S491R mutant (mutation at the first residue following the A9 motif) showed elevated overall activity compared to the wild-type protein. Our results suggest that the A9 core sequence plays a role in the second reaction step, in which it could serve as a "clip" for the proper positioning of residues important for the interaction with the T-domain, and/or stabilisation of the thioester-forming conformation.
authors.affiliation: Department of Chemistry, Faculty of Science, University of Split, N. Tesle 12, Split, Croatia. viljemka@pmfst.hr
authors.forename: Viljemka
authors.initials: V
authors.lastname: Bučević-Popović
authors.forename: Matilda
authors.initials: M
authors.lastname: Sprung
authors.forename: Barbara
authors.initials: B
authors.lastname: Soldo
authors.forename: Maja
authors.initials: M
authors.lastname: Pavela-Vrančič
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D046908:Peptide Biosynthesis, Nucleic Acid-Independent; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D016415:Sequence Alignment
chemical_list: D010453:Peptide Synthases; C035180:tyrocidine synthetase