Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states

Abstract

Ketosynthases (KSs) catalyse essential carbon–carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 12 C atoms, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-linking methods to capture and elucidate conformational transitions accompanying KS-mediated catalysis at near-atomic resolution.

Masked cerulenin enables a dual-site selective protein crosslink

“…5a) also reveal details of the AcpP·FabB interaction as well as providing further support for its gated mechanism. 15,36,37 As shown in ESI Fig. S17,† the FabB = AcpP crosslinked structure revealed the electrostatic interactions that FabB (positively charged) and AcpP (negatively charged) formed within R62, K63, R66, R124, K127, Y132 of FabB and Q14, D35, D38, E47 of AcpP.…”

“…This was veried by the coordination between the C1 amide carbonyl and H298 and H333 in the FabB His-His-Cys catalytic triad. 20,36 To verify if crosslinker 1c mimics the cerulenin binding mode as it was designed, we overlayed the structures of cerulenin bound FabB (PDB: 1FJ8), FabB crosslinked with C12aBr-crypto-AcpP (PDB: 6OKC), and FabB crosslinked with (2R,3S)-1c-crypto-AcpP (PDB: 8MSM). Crosslinker (2R,3S)-1c trapped the crosslinked complex in its catalytic relevant state sharing similar substrateprotein interactions (C163, V270, H298, T300, T302, H333, and F392) with the previously reported structures (ESI Fig.…”

Mitochondrial Acyl Carrier Protein of Leishmania major Displays Features Distinct from the Canonical Type II ACP

“…Single-site mutations of ACP were introduced using a sitedirected mutagenesis approach and purified analogously to the wild-type protein. For NMR measurements, uniformly labeled [ 1 H, 15 N, 13 C] apo-ACP was prepared by growing E. coli Bl21(DE3) cells (transformed with the desired plasmid) in minimal (M9) media containing 15 N NH 4 Cl (1g/l), 13 C glucose (2g/l), vitamins, etc.…”

“…NMR Data Acquisition. NMR samples comprised uniformly labeled [ 1 H, 15 N, 13 C] protein (0.5−1 mM) in 20 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl, 2 mM DTT, 0.5% sodium azide, 90% H 2 O, and 10% D 2 O. Two-and three-dimensional NMR experiments, viz. 1 H 15 N HSQC, CBCAcoNH, CCcoNH, and HNCA experiments, were performed on a Bruker Avance III 700 MHz NMR spectrometer, installed at the National Institute of Immunology, New Delhi, India, equipped with a tunable multinuclear triple resonance TCI/TXI probe.…”

“…In this covalently associated assembly, the acyl chain is partially protected in the hydrophobic cavity of the acyl carrier protein domain. , The pathway has been well characterized in mammals − and a few microorganisms like Mycobacterium tuberculosis, Saccharomyces cerevisiae, , and Candida albicans . The type II pathway is found mostly in prokaryotes, e.g., Escherichia coli, − Bacillus subtilis, M. tuberculosis, , etc.…”