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

Abstract

At the center of many complex biosynthetic pathways,t he acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides.C arrier proteins covalently tether their cargo via at hioester linkage to ap hosphopantetheine cofactor.D ue to the labile nature of this linkage,chemoenzymatic methods have been developed that involve replacement of the thioester with amore stable amide or ester bond. We explored the importance of the thioester bond to the structure of the carrier protein by using solution NMR spectroscopya nd molecular dynamics simulations.Remarkably,the replacement of sulfur with other heteroatoms results in significant structural changes,t hus suggesting more rigorous selections of isosteric substitutes is needed.Acyl carrier proteins (ACPs) are involved in pathways with metabolic,p harmaceutical, and environmental significance. [1][2][3] Ac lear structural understanding of ACPs and their interactions with partner enzymes is essential to furthering metabolic engineering and drug discovery.E fforts in this regard have proven challenging due to transient interactions and the dynamic nature of the ACP, which transports intermediates to multiple enzymes via ac ovalent but labile thioester linkage.T he archetypical carrier protein AcpP,w hich is involved in type II fatty acid biosynthesis in Escherichia coli,h as been well studied, [4,5] but key questions regarding conformational dynamics and interactions between AcpP and partner enzymes remain partially understood. 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. Chemoenzymatic preparations of ACPs bearing crosslinking probes have been used to covalently trap ACP-enzyme complexes. [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. What remains unknown, however, are the dynamics of these interactions throughout ACP-partner protein binding events, including how intermediates attached to the ACPi nfluence protein dynamics and molecular recognition. E. coli AcpP interacts with at least 25 different proteins [9,12,13] and transports acyl intermediates with chain lengths ranging from four to eighteen carbons that assume one of four different boxidation states.G iven the number of proteins with which AcpP interacts and the chemical diversity of the substrates it carries,itislikely that the process by which AcpP delivers its cargo (chain flipping) is not stochastic but rather regulated by substrate-influenced protein-protein interactions. [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 ...

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity

“…This results in a nonhydrolyzable substrate analog that is still tethered to the PPant via a C-S bond and has a very similar structure to the real substrate (Supplementary Fig. 9), circumventing issues encountered with other stabilization strategies 34 . To obtain crystals of the PCP 2 -C 3 construct with this substrate analog (hereafter referred to as Gly stab ) bound, we again used Sfp to attach PPant-Gly stab to the PCP domain.…”

“…6b, see Supplementary Figs. [30][31][32][33][34][35]. Next, we tested a simplified benzoic acid (BA)-D-Arg-Gly donor substrate in these assays, which showed acceptable levels of conversion (61%) and hence we retained this simplified substrate for all subsequent assays.…”

Understanding condensation domain selectivity in non-ribosomal peptide biosynthesis: structural characterization of the acceptor bound state

“…This results in a non-hydrolyzable substrate analogue that is still tethered to the PPant via a C-S bond and has a very similar structure to the real substrate (SI Figure S9), circumventing issues encountered with other stabilization strategies. 34 To obtain crystals of the PCP 2 -C 3 construct with this substrate analogue (hereafter referred to as Gly stab ) bound, we again used Sfp to attach PPant-Gly stab to the PCP domain. This construct was then crystallized as previously, resulting in diffraction to a resolution of 1.9 Å (SI Table S1).…”

Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

“…For example, recent studies have demonstrated that engineering enzyme specificity for a non-native AcpP-dependent enzyme can be accomplished by modifying the PPI residues for improved binding 25 . Even single atom changes in the identity of AcpP-bound cargo have been demonstrated to impart perturbations to the structure of acyl-AcpP 26 , 27 .

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