A KAS-III Heterodimer in Lipstatin Biosynthesis Nondecarboxylatively Condenses C 8 and C 14 Fatty Acyl-CoA Substrates by a Variable Mechanism during the Establishment of a C 22 Aliphatic Skeleton.

β-Ketoacyl-acyl carrier protein (ACP) synthase-III (KAS-III) and its homologs 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 homologs 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 homologs 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 C 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 an unidirectional nucleophilic substitution. Uncovering LstAB catalysis draws attention to thiolase-fold proteins that are non-canonical in both active form and catalytic reaction/mechanism. LstAB Homologs are widespread in bacteria and remain to be functionally assigned, generating great interest in their corresponding products and associated biological functions.