Reprogramming acyl carrier protein interactions of an Acyl-CoA promiscuous trans-acyltransferase

Abstract

Protein interactions between acyl carrier proteins (ACPs) and trans-acting acyltransferase domains (trans-ATs) are critical for regioselective extender unit installation by many polyketide synthases, yet little is known regarding the specificity of these interactions, particularly for trans-ATs with unusual extender unit specificities. Currently, the best-studied trans-AT with nonmalonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed an assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a noncognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions and for engineering modular polyketide synthases to produce analogs.

Figure 1

Substrate specificity of trans-AT’s. (A) Examples of polyketides biosynthesized via trans-AT’s. The contribution of each trans-AT, and the corresponding substrate are shown color-coded. (B) Examples of acyl-thioester substrates for trans-AT’s.

Figure 2

Biosynthesis of kirromycin. The putative biosynthetic contribution of KirCI (malonyl-CoA, red) and KirCII (ethylmalonyl-CoA, purple) to the final structure of kirromycin is highlighted.

Figure 3

Cycloaddition assay for KirCII:ACP interactions. (A) KirCII-catalyzed trans-acylation and subsequent cycloaddition. (B) In-gel fluorescence detection of a series of KirCII control assays. Coomassie Blue image shows ACP loading in each reaction. (C) Fluorescence intensity of labeled ACP5_Kir in a series of control reactions using various trans-ATs or no enzyme. (D) Fluorescence intensity of KirCII-labeled ACP5_Kir as a function of time. (E) Fluorescence intensity of labeled ACP5_Kir as a function of KirCII concentration. Error bars represent the standard deviation of the mean (n=3)

Figure 4

ACP specificity of KirCII. Activity of 3 μM KirCII with 30 μM each kirromycin ACP and 300 μM AzEM-CoA was determined by the cycloaddition assay. Error bars represent the standard deviation of the mean (n=3).

Figure 5

Mapping the KirCII:ACP interaction epitope by alanine scanning mutagenesis. (A) Trans-acylation rates of holo-ACP5_Kir alanine mutants with KirCII. Rates are expressed as a percentage of the activity with wild-type ACP5_Kir. Wild-type positions that were Ala/Gly were not mutated. Mutants that displayed <20% the activity of the wild-type holo-ACP5_Kir are highlighted red. (B) Trans-acylation activities of ACP5_Kir alanine mutants mapped onto an ACP5_Kir homology model ribbon diagram (left) and its computed surface (right). Red, <20% activity; green, 20–80% activity; yellow, >80% activity; grey, not determined. Error bars represent the standard deviation of the mean (n=3).

Figure 6

Docking model of the KirCII:ACP5_Kir interaction. (A) A homology model for ACP5_Kir was used as a ligand to dock with a homology model for KirCII. See Experimental Procedures for details regarding generation of the homology models. KirCII large subdomain is shown in light orange, the small subdomain of KirCII is shown in light pink, KirCII linker domain is green, and ACP5_Kir is shown in teal. The H_II’ portion of L_I of ACP5_Kir is highlighted red. Phosphopantetheinylation site of ACP5_Kir and the KirCII catalytic Ser are shown as spheres. For clarity, not every highlighted residue is labeled. (B) Mutational analysis of KirCII putative interface residues. Error bars represent the standard deviation of the mean (n=3). (C) Electrostatic surface potential maps of KirCII and ACP5_Kir are calculated using the PDB2PQR server. Colors range from blue (positive) to white (neutral) to red (negative). Four key electrostatic contacts are shown boxed, the KirCII contacts are A (D98), B (R410) C (R409), and D (E245), while those of ACP5_Kir are A′ (R51), B′ (D60), C′ (D64), and D′ (R69). The asterisk indicates the position of the KirCII active site Ser and the ACP5_Kir phosphopantetheinylation site. The surfaces are represented so that if ACP5_Kir is rotated 180°, the letters from each surface would match.

Figure 7

Probing the ACP:KirCII interaction epitope by mutagenesis of a non-cognate ACP. (A) Sequence logo for the kirromycin ACP’s. Shown in grey and orange, respectively, are the amino acids at selected positions of ACP5_Kir and ACP10_Kir. Asterisk indicates the phosphopantetheinylation site. Positions discussed in the text are highlighted. Boundaries between each secondary structure element are also shown. For brevity, N- and C-terminal loop regions (L₀ and L_III) are not shown. L₀ is the region preceding H_I and L_III is the region following H_III. (B) Scheme showing contribution of ACP5_Kir and ACP10_Kir to each chimera or mutant (left). Activities of WT, chimeric, and mutant kirromycin ACP’s with KirCII (right). Rates are expressed as a percentage of the activity with WT holo-ACP5_Kir. (C) Activities of WT and mutant ACP6_DEBS with KirCII, expressed as a percentage of the activity with WT holo-ACP5_Kir. See Experimental Procedures for reaction and assay conditions. Error bars represent the standard deviation of the mean (n=3).