Differentiating carrier protein interactions in biosynthetic pathways using dapoxyl solvatochromism

Abstract

Carrier protein-dependent synthases are ubiquitous enzymes involved both in primary and secondary metabolism. Biocatalysis within these synthases is governed by key interactions between the carrier protein, substrate, and partner enzymes. The weak and transient nature of these interactions has rendered them difficult to study. Here we develop a useful fluorescent solvatochromic probe, dapoxyl-pantetheinamide, to monitor and quantify carrier protein interactions in vitro. Upon loading with target carrier proteins, we observe dramatic shifts in fluorescence emission wavelength and intensity and further demonstrate that this tool has the potential to be applied across numerous biosynthetic pathways. The environmental sensitivity of this probe allows rapid characterization of carrier protein interactions, with the ability to quantitatively determine inhibition of protein-protein interactions. We anticipate future application of these probes for inhibitor screening and in vivo characterization.

Fig. 1. Design and characterization of dapoxyl-pantetheinamide. (a) Dapoxyl-pantetheinamide is a bifunctional probe consisting of a solvatochromic fluorophore and CP-specific substrate analogue. (b) Dapoxyl-pantetheinamide dissolved in solvent mixtures ranging from pure CH₂Cl₂ to pure MeOH. Visualization under 365 nm UV light.

Scheme 1. Synthesis of dapoxyl-pantetheinamide (1) from 4-dimethylaminoacetophenone (2) and p-anisaldehyde acetal protected pantetheinamine 10.

Scheme 2. One-pot chemoenzymatic probe-loading reaction. This process involves two discrete steps run in tandem. The first serves to convert the dapoxyl-pantetheinamide 1 into the corresponding coenzyme A analogue 1a, which is then loaded on to an apo-carrier protein (CP) to form a fluorescent crypto-CP 1b. The most fluorescent state occurs upon protein loading in 1b.

Fig. 2. Application to E. coli AcpP. (a) Reaction mixtures of 1, 1a and loading onto AcpP as crypto-1b-AcpP (Scheme 2). (b) 15% urea-PAGE analysis of reaction mixtures visualized by Coomassie Blue stain (left) and 365 nm UV light (right). (c) Fluorescence emission spectra of reaction mixtures. Each reaction is denoted by colored squares which match the color of the plots in (c).

Fig. 3. Dapoxyl fluorescence profiling of various CPs. (a) Reaction mixtures of 1, 1a and loading onto CPs as their crypto-1b-CP state (Scheme 2). 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, enterobactin nonribosomal peptide synthase peptidyl carrier protein; PltL, pyoluteorin nonribosomal peptide synthase peptidyl carrier protein. (b) 15% urea-PAGE analysis of reaction mixtures visualized by Coomassie Blue stain (left) and 365 nm UV light (right). (c) Fluorescence emission spectra of reaction mixtures. Each reaction is denoted by colored squares which match the color of the plots in (c).

Fig. 4. (a) Type II fatty acid biosynthesis in E. coli. The fatty acyl substrate is tethered to carrier protein AcpP via a thioester linkage on the terminal thiol of Ser36-4′-phosphopantetheine. AcpP shuttles the growing fatty acyl substrate to various partner enzymes to catalyze biosynthesis. Partner enzymes include malonyl-CoA-AcpP acyltransferase FabD; ketosynthases FabH, FabF, and FabB; ketoreductase FabG; dehydratases FabZ and FabA; and enoyl reductase FabI. (b) Reaction mixtures of crypto-1b-AcpP (50 μM) loading reactions with an added 1 : 1 equivalent of fatty acid synthase partner protein visualized with 365 nm UV. (c) Fluorescence emission spectra of crypto-1b-AcpP interacting with partner proteins. Each mixture in (b) is denoted by colored squares which match the color of the plots in (c).

Fig. 5. (a) Loading reactions of crypto-1b-AcpP (50 μM) with added FabB (50 μM) and cerulenin (various concentrations) visualized with 365 nm UV. (b) Fluorescence emission spectra of crypto-1b-AcpP (50 μM) loading reactions with added FabB (50 μM) and cerulenin (various concentrations). (c) Dose response curve of maximum AcpP–FabB fluorescence emission in response to increasing concentrations of cerulenin. (d) Loading reactions of crypto-1b-AcpP (50 μM) loading reactions with added FabF (50 μM) and cerulenin (various concentrations) visualized with 365 nm UV. (e) Fluorescence emission spectra of crypto-1b-AcpP (50 μM) loading reactions with added FabF (50 μM) and cerulenin (various concentrations). (f) Dose response curve of maximum AcpP–FabF fluorescence emission in response to increasing concentrations of cerulenin.