Chasing acyl carrier protein through a catalytic cycle of lipid A production

Abstract

Summary Acyl-carrier-protein (ACP) represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon-chains in vivo. Notably, type II ACPs serve as a crucial interaction hub within primary cellular metabolism1 by communicating transiently between partner enzymes of the numerous biosynthetic pathways2,3. However, the highly transient nature of such interactions and the inherent conformational mobility of ACP2 have stymied previous attempts to structurally visualize ACP tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but are also of therapeutic value. For example, ACP is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides (LPS) in Gram-negative microorganisms, which is required for their growth and survival4,5 and is an activator of the mammalian host's immune system6,7, thus emerging as an important therapeutic target8-10. During lipid A synthesis (Raetz Pathway), ACP shuttles acyl-intermediates linked to its prosthetic 4′-phosphopantetheine group (4′-PPT)2 among four acyltransferases, including LpxD11. Here we report the crystal structures of three forms of Escherichia coli ACP engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4′-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release.

Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism

“…S4D). That ACP interaction with proteins is primarily driven by electrostatics is well established (26,27). Indeed, we found that by substituting these residues, the observed substrate inhibition was attenuated, and the remaining AbLpxM activity exhibited a V max of ∼0.6·min −1 , directly supporting our model (SI Appendix, Fig.…”

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

“…However, the binding of FITC-RJPXD33⌬6-COOH (36 Ϯ 3.5 M) did not show increased affinity to LpxD as it did to LpxA ( Table 2) and reflects the fact that the role of hydrocarbon ruler in EcLpxD is fulfilled by Met-290 instead of a basic histidine residue as with LpxA (Fig. 7D) (10,35).…”

“…The Tyr-4 and Leu-6 side chains of RJPXD33 occupy the O-channel, which is postulated to be the binding site for the ester-linked acyl group of substrate UDP-3-O-acyl-GlcN. The six C-terminal residues of RJPXD33 would potentially extend into a binding groove near the C-terminal ␣-helical domain of EcLpxD, which is not present in EcLpxA and has been identified as the ACP binding domain (35). This would explain the role of the six C-terminal residues with respect to the increased affinity of RJPXD33 for LpxD when compared with LpxA as well as the greater loss in affinity between full-length peptide and the C-terminal ⌬6 peptide for LpxD over LpxA.…”

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

“…S6). We compared the surface features of other ACPs in complexes with cognate enzymes (24)(25)(26)(27)(28)(29) to ACP D (Fig. S6).…”