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Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB inEscherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible forE. coliFAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

 

Abstract. Ice-nucleating particles (INPs) represent a rare subset of aerosol particlesthat initiate cloud droplet freezing at temperatures above the homogenousfreezing point of water (−38 ∘C). Considering that the oceancovers 71 % of the Earth's surface and represents a large potential sourceof INPs, it is imperative that the identities, properties and relativeemissions of ocean INPs become better understood. However, the specificunderlying drivers of marine INP emissions remain largely unknown due tolimited observations and the challenges associated with isolating rare INPs. Bygenerating isolated nascent sea spray aerosol (SSA) over a range ofbiological conditions, mesocosm studies have shown that marine microbes cancontribute to INPs. Here, we identify 14 (30 %) cultivable halotolerantice-nucleating microbes and fungi among 47 total isolates recovered fromprecipitation and aerosol samples collected in coastal air in southernCalifornia. Ice-nucleating (IN) isolates collected in coastal air were nucleated ice fromextremely warm to moderate freezing temperatures (−2.3 to −18 ∘C). While some Gammaproteobacteria and fungi are known to nucleate ice attemperatures as high as −2 ∘C, Brevibacterium sp. is the first Actinobacteriafound to be capable of ice nucleation at a relatively high freezingtemperature (−2.3 ∘C). Air mass trajectory analysis demonstratesthat marine aerosol sources were dominant during all sampling periods, andphylogenetic analysis indicates that at least 2 of the 14 IN isolates areclosely related to marine taxa. Moreover, results from cell-washingexperiments demonstrate that most IN isolates maintained freezing activityin the absence of nutrients and cell growth media. This study supportsprevious studies that implicated microbes as a potential source of marineINPs, and it additionally demonstrates links between precipitation, marineaerosol and IN microbes. 

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr,Microbiol. Rev.57, 522–542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al.,Chem. Biol.17, 776–785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein–protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart,ChemBioChem16, 528–547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein–protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.