title: Yersiniabactin synthetase: probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HWMP2 subunit.
abstract: The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Miller
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000591:Amino Acid Isomerases / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003066:Coenzyme A Ligases; D004789:Enzyme Activation / Q000235:genetics; D033862:Iron-Binding Proteins; D008970:Molecular Weight; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D010636:Phenols; D017434:Protein Structure, Tertiary / Q000235:genetics; D012694:Serine / Q000235:genetics; D017262:Siderophores / Q000096:biosynthesis*; D013379:Substrate Specificity / Q000235:genetics; D013844:Thiazoles; D015010:Yersinia pestis / Q000201:enzymology* / Q000235:genetics
chemical_list: D000213:Acyl Carrier Protein; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D033862:Iron-Binding Proteins; D033902:Periplasmic Binding Proteins; D010636:Phenols; D017262:Siderophores; D013844:Thiazoles; C104398:yersiniabactin; D012694:Serine; D000591:Amino Acid Isomerases; D003066:Coenzyme A Ligases; C408477:YbtE protein, Yersinia pestis; D010453:Peptide Synthases; D000409:Alanine