Versatility of Acyl-Acyl Carrier Protein Synthetases

Abstract

The acyl carrier protein (ACP) requires post-translational modification with a 4’-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-acyl carrier protein synthetases (AasSs) from different organisms are able to load even, odd and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.

Exogenous fatty acid metabolism in bacteria

“…The expression of Vh Aas in E. coli allows the elongation of medium chain exogenous fatty acids and their subsequent incorporation into the membrane phospholipids [35,51]. Vh Aas accepts a broad array of acyl chains including unnatural fatty acid analogues, and has been extensively used as a chemical biology tool to generate unnatural acyl-ACP molecules [52]. However, the function of Vh Aas in the parent organism has been poorly characterized.…”

Unsaturated Fatty Acid Synthesis in the Gastric Pathogen Helicobacter pylori Proceeds via a Backtracking Mechanism

“…However, expression of Vibrio harveyi acyl-ACP synthetase (AasS) allows exogenous fatty acids to enter the fatty acid synthetic pathway (Beld et al, 2014; Bi et al, 2013; Jiang et al, 2010). Hence, we transformed the E. coli fabA deletion mutant strain HW8 (which lacks the ability to convert UFA moieties to their cyclopropane derivatives) with plasmids encoding FabX and AasS and labeled the cells with [1- 14 C]-C8, -C10 or -C12 saturated fatty acids.…”

Modifying the Thioester Linkage Affects the Structure of the Acyl Carrier Protein

“…Methods to capture ACP-partner proteins interactions have been developed, and often require the preparation of acyl-ACPs in which the naturally occurring thioester linkage has been replaced with am ore stable amide or ester bond. [16] Substrate loading was monitored with conformationally sensitive urea polyacrylamide gel electrophoresis (PAGE). [6][7][8] Using this strategy,crystal structures of E. coli fatty acid ACP( AcpP) crosslinked to FabA, [9] FabB, [10] and FabZ [11] have been determined, which reveal discrete molec-ular interactions that mediate AcpP binding to these enzymes.…”

“…[14] Due the geometric and electronic differences between thioester, ester,a nd amide linkages,e valuation of such modifications is necessary.W es ought to determine how the structure of AcpP is affected by the chemical nature of the substrate-AcpP linkage.I nt his study we used solution-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) to evaluate the influence of thioester, ester,and amide linkages upon the structure of AcpP and its interactions with ap artner enzyme.First, solution NMR was used to compare the structures of variants of octanoyl-AcpP (C8-AcpP) featuring either athioester (C8-S-AcpP), amide (C8-NH-AcpP), or ester (C8-O-AcpP) linkage.T he thioacyl-AcpP analogues were synthesized using achemoenzymatic "one-pot" method, [15] while the native thioacyl-AcpP was enzymatically synthesized with the enzyme VhAasS (Scheme 1). [16] Substrate loading was monitored with conformationally sensitive urea polyacrylamide gel electrophoresis (PAGE). Linkage-based differences in gel migration were observed ( Figure S1B in the Supporting Information).…”