Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Substrate Sequestration and Chain Flipping in Human Mitochondrial Acyl Carrier Protein.

Outside of their involvement in energy production, mitochondria play a critical role for the cell through their access to a discrete pathway for fatty acid biosynthesis. Despite decades of study in bacterial fatty acid synthases (the putative evolutionary mitochondrial precursor), our understanding of human mitochondrial fatty acid biosynthesis remains incomplete. In particular, the role of the key carrier protein, human mitochondrial acyl carrier protein (mACP), which shuttles the substrate intermediates through the pathway, has not been well-studied in part due to challenges in protein expression and purification. Herein, we report a reliable method for recombinant Escherichia coli expression and purification of mACP. Fundamental characteristics, including substrate sequestration and chain-flipping activity, are demonstrated in mACP using solvatochromic response. This study provides an efficient approach toward understanding the fundamental protein-protein interactions of mACP and its partner proteins, ultimately leading to a molecular understanding of human mitochondrial diseases such as mitochondrial fatty acid oxidation deficiencies.

Masked cerulenin enables a dual-site selective protein crosslink.

Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases. We also describe two new classes of silylcyanohydrin and silylhemiaminal masked crosslinking probes that serve as new tools for activity and structure studies of these biosynthetic pathways.

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

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.

Enzymology of standalone elongating ketosynthases.

The ß-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.

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases.

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

Structural basis for selectivity in a highly reducing type II polyketide synthase.

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a ß-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.

Gating mechanism of elongating ß-ketoacyl-ACP synthases.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, ß-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.

Type II fatty acid and polyketide synthases: deciphering protein-protein and protein-substrate interactions.

Covering: up to April 5, 2018 Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional roles. The former are considered primary metabolites that are linear hydrocarbon acids, while the latter are complex aromatic or polyunsaturated secondary metabolites. Though the study of bacterial FAS has benefitted from decades of biochemical and structural investigations, type II PKSs have remained less understood. Here we review the recent approaches to understanding the protein-protein and protein-substrate interactions in these pathways, with an emphasis on recent chemical biology and structural applications. New approaches to the study of FAS have highlighted the critical role of the acyl carrier protein (ACP) with regard to how it stabilizes intermediates through sequestration and selectively delivers cargo to successive enzymes within these iterative pathways, utilizing protein-protein interactions to guide and organize enzymatic timing and specificity. Recent tools that have shown promise in FAS elucidation should find new approaches to studying type II PKS systems in the coming years.