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Probing the Interactions of early polyketide intermediates with the Actinorhodin ACP from S. coelicolor A3(2).

Evans SE, Williams C, Arthur CJ, Płoskoń E, Wattana-amorn P, Cox RJ, Crosby J, Willis CL, Simpson TJ, Crump MP
J Mol Biol 389 511-28 (2009)
Related entries: 2kg6, 2kg8, 2kg9, 2kga, 2kgc, 2kgd

Cited: 31 times
EuropePMC logo PMID: 19361520

Abstract

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 (1)H-(15)N 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 (1)H-(15)N 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.

Reviews citing this publication (7)

  1. The structural role of the carrier protein--active controller or passive carrier. Crosby J, Crump MP. Nat Prod Rep 29 1111-1137 (2012)
  2. The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal. Cronan JE. Biochem J 460 157-163 (2014)
  3. New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis. Kittilä T, Mollo A, Charkoudian LK, Cryle MJ. Angew Chem Int Ed Engl 55 9834-9840 (2016)
  4. Structural analysis of protein-protein interactions in type I polyketide synthases. Xu W, Qiao K, Tang Y. Crit Rev Biochem Mol Biol 48 98-122 (2013)
  5. Type II fatty acid and polyketide synthases: deciphering protein-protein and protein-substrate interactions. Chen A, Re RN, Burkart MD. Nat Prod Rep 35 1029-1045 (2018)
  6. Enzymology of standalone elongating ketosynthases. Chen A, Jiang Z, Burkart MD. Chem Sci 13 4225-4238 (2022)
  7. Probing the structure and function of acyl carrier proteins to unlock the strategic redesign of type II polyketide biosynthetic pathways. Sulpizio A, Crawford CEW, Koweek RS, Charkoudian LK. J Biol Chem 296 100328 (2021)

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  2. Understanding Programming of Fungal Iterative Polyketide Synthases: The Biochemical Basis for Regioselectivity by the Methyltransferase Domain in the Lovastatin Megasynthase. Cacho RA, Thuss J, Xu W, Sanichar R, Gao Z, Nguyen A, Vederas JC, Tang Y. J Am Chem Soc 137 15688-15691 (2015)
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  4. Recognition of intermediate functionality by acyl carrier protein over a complete cycle of fatty acid biosynthesis. Płoskoń E, Arthur CJ, Kanari AL, Wattana-amorn P, Williams C, Crosby J, Simpson TJ, Willis CL, Crump MP. Chem Biol 17 776-785 (2010)
  5. A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis. Haines AS, Dong X, Song Z, Farmer R, Williams C, Hothersall J, Płoskoń E, Wattana-Amorn P, Stephens ER, Yamada E, Gurney R, Takebayashi Y, Masschelein J, Cox RJ, Lavigne R, Willis CL, Simpson TJ, Crosby J, Winn PJ, Thomas CM, Crump MP. Nat Chem Biol 9 685-692 (2013)
  6. Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase. Charkoudian LK, Liu CW, Capone S, Kapur S, Cane DE, Togni A, Seebach D, Khosla C. Protein Sci 20 1244-1255 (2011)
  7. Reversible labeling of native and fusion-protein motifs. Kosa NM, Haushalter RW, Smith AR, Burkart MD. Nat Methods 9 981-984 (2012)
  8. Structural and biochemical characterization of ZhuI aromatase/cyclase from the R1128 polyketide pathway. Ames BD, Lee MY, Moody C, Zhang W, Tang Y, Tsai SC. Biochemistry 50 8392-8406 (2011)
  9. Letter Binding and "pKa" modulation of a polycyclic substrate analogue in a type II polyketide acyl carrier protein. Haushalter RW, Filipp FV, Ko KS, Yu R, Opella SJ, Burkart MD. ACS Chem Biol 6 413-418 (2011)
  10. Characterization of molecular interactions between ACP and halogenase domains in the Curacin A polyketide synthase. Busche A, Gottstein D, Hein C, Ripin N, Pader I, Tufar P, Eisman EB, Gu L, Walsh CT, Sherman DH, Löhr F, Güntert P, Dötsch V. ACS Chem Biol 7 378-386 (2012)
  11. Probing the phosphopantetheine arm conformations of acyl carrier proteins using vibrational spectroscopy. Johnson MN, Londergan CH, Charkoudian LK. J Am Chem Soc 136 11240-11243 (2014)
  12. Modeling linear and cyclic PKS intermediates through atom replacement. Shakya G, Rivera H, Lee DJ, Jaremko MJ, La Clair JJ, Fox DT, Haushalter RW, Schaub AJ, Bruegger J, Barajas JF, White AR, Kaur P, Gwozdziowski ER, Wong F, Tsai SC, Burkart MD. J Am Chem Soc 136 16792-16799 (2014)
  13. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase. Vance S, Tkachenko O, Thomas B, Bassuni M, Hong H, Nietlispach D, Broadhurst W. Biochem J 473 1097-1110 (2016)
  14. Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury. Vercelli A, Biggi S, Sclip A, Repetto IE, Cimini S, Falleroni F, Tomasi S, Monti R, Tonna N, Morelli F, Grande V, Stravalaci M, Biasini E, Marin O, Bianco F, di Marino D, Borsello T. Cell Death Dis 6 e1854 (2015)
  15. A nuclear magnetic resonance method for probing molecular influences of substrate loading in nonribosomal peptide synthetase carrier proteins. Goodrich AC, Frueh DP. Biochemistry 54 1154-1156 (2015)
  16. Phosphopantetheinylation and specificity of acyl carrier proteins in the mupirocin biosynthetic cluster. Shields JA, Rahman AS, Arthur CJ, Crosby J, Hothersall J, Simpson TJ, Thomas CM. Chembiochem 11 248-255 (2010)
  17. Recognition of extended linear and cyclised polyketide mimics by a type II acyl carrier protein. Dong X, Bailey CD, Williams C, Crosby J, Simpson TJ, Willis CL, Crump MP. Chem Sci 7 1779-1785 (2016)
  18. Crystal structure of MBP-PigG fusion protein and the essential function of PigG in the prodigiosin biosynthetic pathway in Serratia marcescens FS14. Zhang F, Wei Q, Tong H, Xu D, Wang W, Ran T. Int J Biol Macromol 99 394-400 (2017)
  19. Solution structure of 4'-phosphopantetheine - GmACP3 from Geobacter metallireducens: a specialized acyl carrier protein with atypical structural features and a putative role in lipopolysaccharide biosynthesis. Ramelot TA, Smola MJ, Lee HW, Ciccosanti C, Hamilton K, Acton TB, Xiao R, Everett JK, Prestegard JH, Montelione GT, Kennedy MA. Biochemistry 50 1442-1453 (2011)
  20. Structure, function and dynamics in acyl carrier proteins. Farmer R, Thomas CM, Winn PJ. PLoS One 14 e0219435 (2019)
  21. A Light-Activated Acyl Carrier Protein "Trap" for Intermediate Capture in Type II Iterative Polyketide Biocatalysis. Kilgour SL, Kilgour DPA, Prasongpholchai P, O'Connor PB, Tosin M. Chemistry (2019)
  22. Tracking carrier protein motions with Raman spectroscopy. Epstein SC, Huff AR, Winesett ES, Londergan CH, Charkoudian LK. Nat Commun 10 2227 (2019)
  23. A Protein Interaction Map of the Kalimantacin Biosynthesis Assembly Line. Uytterhoeven B, Lathouwers T, Voet M, Michiels CW, Lavigne R. Front Microbiol 7 1726 (2016)
  24. Path to Actinorhodin: Regio- and Stereoselective Ketone Reduction by a Type II Polyketide Ketoreductase Revealed in Atomistic Detail. Serapian SA, Crosby J, Crump MP, van der Kamp MW. JACS Au 2 972-984 (2022)


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