TY - JOUR

T1 - Probing the Interactions of Early Polyketide Intermediates with the Actinorhodin ACP from S. coelicolor A3(2)

AU - Evans, Simon E.

AU - Williams, Christopher

AU - Arthur, Christopher J.

AU - Płoskoń, Eliza

AU - Wattana-amorn, Pakorn

AU - Cox, Russell J.

AU - Crosby, John

AU - Willis, Christine L.

AU - Simpson, Thomas J.

AU - Crump, Matthew P.

PY - 2009/6/12

Y1 - 2009/6/12

N2 - Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. Seven thiol ester and thiol ether derivatives of the actinorhodin (act) PKS ACP from Streptomyces coelicolor have been prepared and structurally characterised by NMR to gain insight into ACP-intermediate interactions. Holo ACP synthase has been used to prepare early-stage ACP intermediates of polyketide biosynthesis (holo ACP, acetyl ACP, and malonyl ACP) from the respective coenzyme A derivatives. A synthetic route to stabilised thiol ether ACPs was developed and applied to the preparation of stable 3-oxobutyl and 3,5-dioxohexyl ACP as diketide and triketide analogues. No interaction between the protein and the acyl phosphopantetheine moieties of acetyl, malonyl, or 3-oxobutyl ACP was detected. Analysis of 1H-15N heteronuclear single quantum coherence and nuclear Overhauser enhancement spectroscopy spectra for the triketide ACP revealed exchange between a major ('Tri', 85%) and a minor protein conformer in which the polyketide interacts with the protein ('Tri*', 15%). Act ACP was also derivatised with butyryl, hexanoyl, and octanoyl groups. The corresponding NMR spectra showed large chemical shift perturbations centred on helices II and III, indicative of acyl chain binding and significant structural rearrangement. Unexpectedly, butyryl act ACP showed almost identical backbone 1H-15N chemical shifts to Tri*, suggesting comparable structural changes that might provide insight into the structurally uncharacterised polyketide bound form. Furthermore, butyryl ACP itself underwent slow conformational exchange with a second minor conformer (But*) with almost identical backbone chemical shifts to octanoyl act ACP. High-resolution NMR structures of these acylated forms revealed that act ACP was able to undergo dramatic conformational changes that exceed those seen in FAS ACPs. When compared to E. coli FAS ACP, the substrate binding pocket of the act PKS ACP has three specific amino acid substitutions (Thr39/Leu45, Ala68/Leu74, and Leu42/Thr48) that alter the size, shape, and location of this cavity. These conformational changes may play a role in protein-protein recognition and assist the binding of bulky polyketide intermediates.

AB - Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. Seven thiol ester and thiol ether derivatives of the actinorhodin (act) PKS ACP from Streptomyces coelicolor have been prepared and structurally characterised by NMR to gain insight into ACP-intermediate interactions. Holo ACP synthase has been used to prepare early-stage ACP intermediates of polyketide biosynthesis (holo ACP, acetyl ACP, and malonyl ACP) from the respective coenzyme A derivatives. A synthetic route to stabilised thiol ether ACPs was developed and applied to the preparation of stable 3-oxobutyl and 3,5-dioxohexyl ACP as diketide and triketide analogues. No interaction between the protein and the acyl phosphopantetheine moieties of acetyl, malonyl, or 3-oxobutyl ACP was detected. Analysis of 1H-15N heteronuclear single quantum coherence and nuclear Overhauser enhancement spectroscopy spectra for the triketide ACP revealed exchange between a major ('Tri', 85%) and a minor protein conformer in which the polyketide interacts with the protein ('Tri*', 15%). Act ACP was also derivatised with butyryl, hexanoyl, and octanoyl groups. The corresponding NMR spectra showed large chemical shift perturbations centred on helices II and III, indicative of acyl chain binding and significant structural rearrangement. Unexpectedly, butyryl act ACP showed almost identical backbone 1H-15N chemical shifts to Tri*, suggesting comparable structural changes that might provide insight into the structurally uncharacterised polyketide bound form. Furthermore, butyryl ACP itself underwent slow conformational exchange with a second minor conformer (But*) with almost identical backbone chemical shifts to octanoyl act ACP. High-resolution NMR structures of these acylated forms revealed that act ACP was able to undergo dramatic conformational changes that exceed those seen in FAS ACPs. When compared to E. coli FAS ACP, the substrate binding pocket of the act PKS ACP has three specific amino acid substitutions (Thr39/Leu45, Ala68/Leu74, and Leu42/Thr48) that alter the size, shape, and location of this cavity. These conformational changes may play a role in protein-protein recognition and assist the binding of bulky polyketide intermediates.

KW - acyl carrier protein

KW - conformational switching

KW - NMR structure

KW - polyketide binding

KW - polyketide biosynthesis

UR - http://www.scopus.com/inward/record.url?scp=65649101867&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2009.03.072

DO - 10.1016/j.jmb.2009.03.072

M3 - Article

C2 - 19361520

AN - SCOPUS:65649101867

VL - 389

SP - 511

EP - 528

JO - Journal of molecular biology

JF - Journal of molecular biology

SN - 0022-2836

IS - 3

ER -