A KAS-III Heterodimer in Lipstatin Biosynthesis Nondecarboxylatively Condenses C8 and C14 Fatty Acyl-CoA Substrates by a Variable Mechanism during the Establishment of a C22 Aliphatic Skeleton

β-Ketoacyl-acyl carrier protein synthase-III (KAS-III) and its homologues are thiolase-fold proteins that typically behave as homodimers functioning in diverse thioester-based reactions for C–C, C–O, or C–N bond formation. Here, we report an exception observed in the biosynthesis of lipstatin. During the establishment of the C22 aliphatic skeleton of this β-lactone lipase inhibitor, LstA and LstB, which both are KAS-III homologues but phylogenetically distinct from each other, function together by forming an unusual heterodimer to catalyze a nondecarboxylating Claisen condensation of C8 and C14 fatty acyl-CoA substrates. The resulting C22 α-alkyl β-ketoacid, which is unstable and tends to be spontaneously decarboxylated to a shunt C21 hydrocarbon product, is transformed by the stereoselective β-ketoreductase LstD into a relatively stable C22 α-alkyl β-hydroxyacid for further transformation. LstAB activity tolerates changes in the stereochemistry, saturation degree, and thioester form of both long-chain fatty acyl-CoA substrates. This flexibility, along with the characterization of catalytic residues, benefits our investigations into the individual roles of the two KAS-III homologues in the heterodimer-catalyzed reactions. The large subunit LstA contains a characteristic Cys-His-Asn triad and likely reacts with C8 acyl-CoA to form an acyl-Cys enzyme intermediate. In contrast, the small subunit LstB lacks this triad but possesses a catalytic Glu residue, which can act on the C8 acyl-Cys enzyme intermediate in a substrate-dependent manner, either as a base for Cα deprotonation or as a nucleophile for a Michael-type addition-initiated cascade reaction, to produce an enolate anion for head-to-head assembly with C14 acyl-CoA through a unidirectional nucleophilic substitution. Uncovering LstAB catalysis draws attention to thiolase-fold proteins that are noncanonical in both active form and catalytic reaction/mechanism. LstAB homologues are widespread in bacteria and remain to be functionally assigned, generating great interest in their corresponding products and associated biological functions.