Substrate Promiscuity of the Aryl-ACP Ligase AltC from the Alterochromide

Alterochromides are antibacterial and cytotoxic lipopeptides produced by Pseudoalteromonas , whose biosynthesis is proposed to begin with the loading of p -coumaric acid ( 1 ) onto the acyl carrier protein (ACP) AltB by AltC, an enzyme annotated as an AMP-binding protein. Herein, we report the characterization of AltC as an aryl-ACP ligase by heterologous expression and in vitro reconstitution with AltB, monitored by UPLC-MS. AltC catalyzes the loading of p -coumaric acid ( 1 ) onto holo-AltB and also accepts its brominated analog, 3-bromo-4-hydroxycinnamic acid ( 4 ), as well as 14 additional cinnamic acid analogs functionalized with hydroxyl, halogen, alkyl, alkoxy, amino and cyano substituents. For electron-poor substrates, the loaded ACP undergoes DTT addition, the extent of which decreases as the ring becomes more electron-rich. Given the broad substrate tolerance, AltC is unlikely to be the selectivity filter for the aryl acid incorporated into (bromo)alterochromides. The promiscuity of AltC also makes it a candidate biocatalyst for preparing non-natural aryl-ACPs and (bromo)alterochromide analogs.