Enzymology of standalone elongating ketosynthases

Abstract

The β-ketoacyl-acyl carrier protein synthase, or ketosynthase (KS), catalyses carbon-carbon bond formation in fatty acid and polyketide biosynthesis via a decarboxylative Claisen-like condensation. In prokaryotes, standalone elongating KSs interact with the acyl carrier protein (ACP) which shuttles substrates to each partner enzyme in the elongation cycle for catalysis. Despite ongoing research for more than 50 years since KS was first identified in E. coli, the complex mechanism of KSs continues to be unravelled, including recent understanding of gating motifs, KS-ACP interactions, substrate recognition and delivery, and roles in unsaturated fatty acid biosynthesis. In this review, we summarize the latest studies, primarily conducted through structural biology and molecular probe design, that shed light on the emerging enzymology of standalone elongating KSs.

Fig. 1. KS catalysis and mechanism-based crosslinkers: (a) transition of the substrate from the ACP pocket to the KS pocket is described as a chain-flipping event. (b) The ping–pong mechanism of KS catalysis begins with the transacylation half-reaction to generate acyl-KS followed by the condensation half-reaction that synthesizes β-ketoacyl-ACP via decarboxylative condensation. (c) The mechanism-based crosslinkers, α-bromo and Cl-acrylate, can trap KS–ACP interaction in the transacylation state and condensation state, respectively. Each leaving group is coloured by red.

Fig. 2. The elongation cycle of type II FAS and PKS: A full cycle of SFA elongation goes through KS-mediated elongation, ketoreduction, dehydration, and enoyl-reduction. The cis-double bond of mono-unsaturated fatty acids comes from the isomerization of (2E)-enoyl-ACP followed by the elongation of FabB (green KS) in a particular cycle. Consecutively skipping the reduction of enoyl-ACP yields polyene intermediates that are elongated by polyene KS–CLF (orange KS), and the elongation catalyzed individually by polyketone KS–CLF (magenta KS) yields chemically flexible intermediates that lead to polycyclic aromatic polyketides (KR = ketoreductase, DH = dehydratase, and ER = enoyl-reductase).

Fig. 3. The gating loops: (a) structure overlay of C12–FabF (PDB ID: 2GFY, blue) in the gate closed conformation and the KS–ACP crosslink complex (PDB ID: 6OKG, green) in the gate open conformation. Movements of F400 on loop 1 and P272 on loop 2 are measured. (b) Surface plot of gate closed FabF (blue) overlaying with the open loop 1 (green) demonstrates the side pocket that accommodates the open loop. (c) G310M mutant blocks the gate from opening. (d) Surface plot of ApeO (PDB ID: 6QSP, orange), a KS from aryl polyene biosynthesis, overlaying with the open loop 1. (e) Surface plot of a polyketone KS, antKS (PDB ID: 6SMO, magenta), overlaying with the open loop 1. The side pocket for the open gate is completely missing. (f) Over a thousand KS gating loop 1 and loop 2 sequences aligned (MUSCLE algorithm) with the consensus sequence shown (left column). The coordination of open loop 1 is depicted on the right. The conserved Asn()–Asp() pair that is essential for the open gate is absent in the polyketone KS. The conserved Leu ( and L286 in antKS) of the polyketone KS clashes into the Phe () of open loop 1. This Leu is instead a conserved Pro in other types of KS.

Fig. 4. Detailed mechanism of KS elongation and the gating loops: FabF, FabB, and polyene KS go through (1)–(7) followed by the product-ACP release for one extension while the polyketone KS goes through the smaller circle (4), (5), (6), and (PK), to keep the growing chain inside the KS pocket. (2) and (7) are considered the substrate delivery and released states, respectively, with the gating loops open and the catalytic oxyanion hole absent. Most depicted states are supported by at least one crystal structure with a representative of each labelled at the bottom-right corner of the box. The amino acid numbering is from EcFabF.

Fig. 5. Gating residues of FabF: overlay of apoFabF (PDB ID: 2GFW, orange), C12–FabF (PDB ID: 2GFY, blue), and the FabF–ACP crosslink structure (PDB ID: 7L4L, light blue). (a) Rotation of F400 triggered by substrate binding creates the malonyl binding pocket. (b) Rotation and translation of I108 when a long acyl-chain (more than 8-carbon) is bound.

Fig. 6. Asymmetric substrate pockets of FabB (PDB ID: 1EK4): E200–Q113 polar contact restricts the pocket size to 12-carbon on the left whereas the pocket with dissociated E200–Q113 on the right accommodates a longer substrate. Q113 from each of the subunit of the homodimer cannot co-occupy the center space, leaving the pockets asymmetric.

Fig. 7. Comparison of the 16-carbon SFA and UFA at the substrate delivery state: overlay of C12–FabF (PDB ID: 2GFY, blue) and FabF–ACP complexes with crosslinkers mimicking C16 (PDB ID: 6OKG, green) and C16:1 (PDB ID: 7L4E, orange), respectively. The cis-double bond of C16:1 redirects the acyl chain to the substrate pocket.

Fig. 8. The PPant binding pocket of FabF: (a) coordination of the two threonine residues paves a hydrophilic path for PPant insertion. (b) Polar contact network of the PPant binding pocket. T270 and S271 belong to gating loop 2.

Fig. 9. Protein–protein interactions of four different KS–ACP interfaces: PPIs are grouped into three regions as depicted in the top graph (ACP helix 4 and the inter-helices loops are not shown for simplicity). PPIs of each region from FabF (PDB ID: 7L4L, blue), FabB (PDB ID: 6OKC, green), polyene KS–CLF (PDB ID: 6KXF, yellow and orange), and polyketone KS–CLF (PDB ID: 6SMP, magenta) are aligned for comparison.

Fig. 10. The chain-flipping mechanism: structure overlay of C10–ACP (PDB ID: 2FAE, green) to ACP helix 2 of the FabF–ACP complex (PDB ID: 7L4L, blue) shows that PPIs initiate chain-flipping. (a) PPIs anchor helix 2, E13, and D56 to twist the 4-helical bundle which shrinks the pocket. (b) Side view of the overlay. Movement of I62 shrinks the pocket and pushes the substrate out.