Yersiniabactin synthetase: Probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HMWP2 subunit

The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosoma l peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three c arrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domain s (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that ge t heterocyclized by the Cy domains to yield a tricyclic hyrdoxyphenylthiazo linylthiazolinyl (HPT-T) chain lodged in thioester linkage to the PCP2 doma in. The interdomain recognition by the Cy1 and Cy2 domains for the three ca rrier proteins was tested using inactivating mutations at the conserved ser ine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, a nd S1977A). These mutant forms of HMWP2 were tested for in trans complement ation by carrier protein fragments: holo-ArCPs (S52A! holo-PCP1 and analogu es (S1439A), and holo-PCP2 and analogues (S1977A!, The S52A mutant tests th e recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S 1439A mutant tests the specificity of the same Cy1 domain for downstream su bstrates presented by distinct PCPs. The S1439A likewise tests the recognit ion of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant anal ogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cas es in trans complementation was successful with the carrier protein fragmen ts, allowing kinetic probes of catalytic efficiency for BCP scaffolds and f or uncoupling of the condensation and heterocyclization functions of Cy1 an d Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acce ptor protein. This work points to the importance of protein-protein interac tions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic effici ency.