Fluorometric Analysis of Carrier-Protein-Dependent Biosynthesis through a Conformationally Sensitive Solvatochromic Pantetheinamide Probe

Abstract

Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP-enzyme interactions.

Figure 1.

Schematic of fluorescent labeling of CPs. A bifunctional probe 1 consisting of a solvatochromic fluorophore (3MC) and pantetheinamide moiety is converted to CoA analog 2 through the action of coenzyme A biosynthetic enzymes. A PPTase loads the CoA analog onto an apo-CP. The resulting crypto-CP 3 attains dramatically altered fluorescent properties due to solvatochromism of the 3MC fluorophore.

Figure 2.

3MC-pantetheinamide labeling of AcpP. (a) Model one-pot chemoenzymatic probe-loading reaction. CoaA, CoaD, and CoaE convert probe 1 into coenzyme A analog 2, which is loaded onto residue Ser36 of apo-AcpP by PPTase Sfp. Sequestration of the 3MC fluorophore within the hydrophobic core of AcpP induces an increase in fluorescence intensity and blue shift. (b) Reaction mixtures of probe 1 loading onto AcpP visualized by 365 nm UV radiation. “CoA loading” refers to CoaA, CoaD, CoaE, and Sfp. (c) 15% urea-PAGE analysis of reaction mixtures visualized by Coomassie Blue stain (left) and 365 nm UV light (right). (d) Reverse-phase HPLC analysis of reaction mixtures R1–R4. Presumed peaks representing apo- and crypto-AcpP are labeled. (e) Fluorescence emission spectra of reaction mixtures R1–R4.

Figure 3.

3MC-pantetheinamide labeling of CPs. “CoA loading” refers to CoaA, CoaD, CoaE, and Sfp. (a) Reaction mixtures of probe 1 loading onto CPs from various selected CP-dependent synthases. (b) 15% urea-PAGE analysis of probe loading reaction mixtures visualized by Coomassie Blue stain (left) and 365 nm UV light (right). AcpP, E. coli fatty acid synthase acyl carrier protein; hACP, human fatty acid synthase acyl carrier protein; DEBS Acp4, 6-deoxyerythronolide B synthase module 4 acyl carrier protein; actACP, actinorhodin polyketide synthase acyl carrier protein; EntB, enter-obactin non-ribosomal peptide synthase peptidyl carrier protein; PltL, pyoluteorin non-ribosomal peptide synthase peptidyl carrier protein. (c) Fluorescence emission spectra of CP probe-loading reaction mixtures.

Figure 4.

Interactions between crypto-PltL loaded with 1 and PltF. (a) Role of PltF as an A domain in pyoluteorin biosynthesis. PltF adenylates proline in an ATP-dependent reaction and transfers the prolyl group to the phosphopantetheine arm of holo-PltL. (b) Hypothesized protein–substrate interaction between crypto-PltL loaded with 1 and PltF. Transfer of the 3MC fluorophore to the active site of PltF results in an increase in fluorescence intensity and blue shift.

Figure 5.

Application of 3MC probe 1 to evaluate CP–partner protein interactions. (a) Fluorescence emission spectra of crypto-PltL loaded with 1 when unbound (no partner protein, noPP), as compared to that in the presence of an equimolar concentration of FabF (noncognate KS), PltF (cognate A domain), or EntE (noncognate A domain). The terms cognate and noncognate are used to distinguish interactions that occur naturally in a given biosynthetic pathway (cognate) or ones that arise from different pathways (noncognate). (b) Differential plot highlighting the differences between each partner protein on crypto-PltL loaded with 1. (c) Fluorescence emission spectra of crypto-AcpP loaded with 1 when unbound (no partner protein, noPP), as compared to that in the presence of an equimolar concentration of FabF (cognate KS), PltF (noncognate A domain), or EntE (noncognate A domain). (d) Differential plot highlighting the differences between each partner protein on crypto-AcpP loaded with 1. (e) Fluorescence emission spectra of crypto-EntB loaded with 1 when unbound (no partner protein, noPP), as compared to that in the presence of an equimolar concentration of FabF (noncognate KS), PltF (noncognate A domain), or EntE (cognate A domain). (f) Differential plot highlighting the differences between each partner protein on crypto-EntB loaded with 1.

Scheme 1.. Modular Synthesis of 3MC-Pantetheinamide Probe 1^a

^aThe 3-methoxychromone fluorophore was synthesized from starting materials 3-dimethylaminophenol (4) and methyl 4-acetoxybenzoate (7). An amide linkage connects the fluorophore to p-anisaldehyde acetal-protected pantothenic acid 13 (synthesis described previously), resulting in probe 1 following deprotection.