Binding and pK_a Modulation of a Polycyclic Substrate Analogue in a Type II Polyketide Acyl Carrier Protein

Abstract

Type II polyketide synthases are biosynthetic enzymatic pathways responsible for the production of complex aromatic natural products with important biological activities. In these systems, biosynthetic intermediates are covalently bound to a small acyl carrier protein that associates with the synthase enzymes and delivers the bound intermediate to each active site. In the closely related fatty acid synthases of bacteria and plants, the acyl carrier protein acts to sequester and protect attached intermediates within its helices. Here we investigate the type II polyketide synthase acyl carrier protein from the actinorhodin biosynthetic pathway and demonstrate its ability to internalize the tricyclic, polar molecule emodic acid. Elucidating the interaction of acyl carrier proteins with bound analogs resembling late-stage intermediates in the actinorhodin pathway could prove valuable in efforts to engineer these systems towards rational design and biosynthesis of novel compounds.

Manipulating Protein–Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins

“…Previously reported, NMR solution structures of PltL in holo - and pyrrolyl forms demonstrated that, like type II fatty acid and polyketide carrier proteins, 2, 27, 28 type II PCPs have the capacity to sequester their substrates. Holo -PigG and holo -PltL have similar structural features.…”

Chemoenzymatic exchange of phosphopantetheine on protein and peptide

“…Evaluated PKS-ACPs included S. coelicolor ActACP , 7 A. parasiticus PksA, 22 G. fujikuroi Pks4, 23 L. majuscula JamC and JamF, 24,25 and P. agglomerans AdmA. 26 Activity in this category was less consistent (Table 1, Supplementary Figures 2–3, 8–9, 11–12), with PfAcpH demonstrating the only activity with crypto - ActACP and Pks4, while all other AcpH except SoAcpH were capable of activity with crypto - PksA and JamC.…”

Resin supported acyl carrier protein labeling strategies

“…2 In 2004, we introduced the post-translational modification of ACP with a phosphopantetheinyltransferase (PPTase) and functional coenzyme A (CoA) analogues in order to attach 4'-phosphopantetheine (PPant) with unique properties, and these tools have been leveraged for enzymology studies and the functionalization of proteins. 3 In addition, we recently introduced the use of an ACP hydrolase (AcpH) to cleave PPant and PPant analogues from ACP as a reversible labeling tool, thus providing a full suite of ACP modifying methodologies. 4 Here we further coordinate this dual enzyme utility as immobilized biocatalytic tools, thereby streamlining their application for protein labeling, unlabeling, and isolation.…”

“…3 These modifications rely on the availability of ACP in the apo - form, a state often limited by the conversion of heterologously expressed apo -ACP to holo -ACP by endogenous PPTases. 9 …”

“…When the apo -ACP is desired, the holo -form of the purified protein goes unused as an expensive side product, a particularly wasteful procedure when isotopes are incorporated into the protein for biophysical studies such as NMR. 3 For these reasons, the conversion of modified ACP to apo - form by AcpH is an attractive purification strategy to provide homogenous products. Here, AcpH cleaves the PPant appendage from both holo- and crypto - (or functionally labeled) ACP, resulting in uniformly apo - protein.…”