RhlA Exhibits Dual Thioesterase and Acyltransferase Activities during Rhamnolipid Biosynthesis

Rhamnolipids (RLs) are a desirable class of biosurfactants originating from Pseudomonas aeruginosa. Rhamnosyltransferase 1 chain A (RhlA) catalyzes the formation of β-3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) to constitute the RL lipid moiety, and the molecular structure of this moiety exerts major impacts on the physiochemical and biological properties of corresponding RLs. However, the catalytic mechanism and sequence-structure–function relationship of RhlA remain elusive. Here, we report the X-ray crystal structure of P. aeruginosa RhlA with an α/β-hydrolase fold and a canonical nucleophile/histidine/acidic catalytic triad. Unexpectedly, free 3-hydroxy fatty acids within a secondary ligand-binding pocket were observed in the crystal of RhlA, which is traditionally considered an acyltransferase that acts only on acyl carrier protein (ACP)-bound substrates. In vitro isotopic labeling, enzyme kinetics experiments, and QM/MM simulations confirmed that free β-hydroxy fatty acids are a reaction intermediate during HAA synthesis. Moreover, first-shell residue mutations that targeted different ligand-binding pockets resulted in distinct modulation patterns for the two acyl chain lengths of HAAs. In conclusion, the revealed biosynthetic mechanism may guide future engineering for the biosynthesis of designer RLs.