A thioester-thiol exchange reaction in Salmonella Invasion acyl carrier protein transfers the 4PP-bound acyl chain to a distal cysteine

Salmonella Typhimurium encodes two functionally distinct acyl carrier proteins: AcpP, involved in fatty acid synthesis, and the invasion acyl carrier protein (IacP), which mediates the acylation of SipB (Salmonella invasion protein B), a modification essential for SipB maturation and efficient host invasion. Despite growing insights, the IacP-dependent machinery governing acyl chain transfer remains largely enigmatic. Therefore, a multidisciplinary approach combining biochemistry and NMR was employed. A D35NIacP mutant (IacPN) that expresses in the apo-form was enzymatically converted into acyl-IacPN intermediates. Unexpectedly, the 1H15N HSQC spectra of the acyl-IacPN intermediates displayed two sets of resonances for several of the amides. The first was a less intense set of peaks, positioned close to the holo-peak, displaying a progressive chemical shift change with increasing acyl-chain length, reminiscent of the 4-phosphopantetheine (4-PP)-bound conformer of type II ACPs. The second, a more intense, acyl-chain-length-independent, previously uncharacterized set of peaks, was clustered near the longer 4PP-bound acyl-chain conformers (C14-). The origin of this new conformer was investigated by generating three cysteine-to-alanine mutants, C16A, C54A, and C69AIacPN. The C54AIacPN mutant displayed a single set of peaks in the acyl-IacPN intermediates, corresponding to the 4PP-bound IacPN conformer, establishing the critical role of Cys 54 in generating the uncharacterized peak and identifying it as a Cys-bound conformer. The acyl-S-carboxyamidomethyl-IacPN intermediates also displayed a single conformer, supporting the role of a cysteine in giving rise to the new peak. Collectively, our studies uncover a unique thioester-thiol exchange reaction in which the 4PP-tethered acyl chain is chemically transferred to a distal cysteine (Cys 54). Given that Cys 54 lies within the SipB interaction interface of IacP, this acyl chain relay process may contribute to IacP-mediated SipB acylation and Salmonella virulence.