Deciphering the structures of trans-AT polyketide synthase megaenzymes Seminar

The modular polyketide synthases (PKSs) – gigantic multienzyme ‘assembly lines’ composed of repeating sets of functional domains − are responsible for the synthesis in bacteria of a wide range of complex polyketide of high medicinal value such as the antibiotic erythromycin and the anti-cancer compound epothilone. An understanding of the structural and mechanistic aspects which underpin interdomain cooperation during biosynthesis by modular PKS is crucial to ongoing efforts to re-engineer these systems for the production of novel compounds for evaluation as drug leads.^1,2 Attempts to solve the structures of PKS modules by X-ray crystallography have been unsuccessful, likely due to the high, inherent flexibility of the multienzymes. We have used an alternative technique, small-angle X-ray scattering (SAXS), in combination with multi-dimensional NMR and homology modelling of individual domains, to characterize the structure of an intact apo module from a trans-acyltransferase (AT) PKS.³

The model system comprises module 5 of subunit VirA of the virginiamycin M1 PKS of Streptomyces virginiae, a target of relevance for its involvement in production of the commercial antibiotic dalfopristin. The investigated module, which includes a ketosynthase (KS) and two consecutive acyl carrier protein (ACP) domains, interacts with a wide range of protein partners, including notably a complex of discrete enzymes responsible for β-methylation of the polyketide, as well as the N-terminus of the downstream PKS protein, VirFG.⁴ Our analysis shows that the homodimeric KS, which is flanked by well-folded linker regions, occupies the center of the module. While the first ACP is located close to the KS, the second is situated at the end of a flexible linker, and mobile. Taken together, these data provide a physical explanation for the functional non-equivalence previously observed for certain tandem ACPs of trans-AT PKS. Furthermore, the overall open shape of the module renders the second ACP highly accessible, which may be critical for its interaction with its multiple in trans catalytic partners. Finally, our data redefine the function of a putative dimerization motif of tandem ACPs as a docking domain, showing that this form of intersubunit communication is conserved between cis- and trans-AT PKS.⁵

 

References

[1] K J Weissman & R Müller (2008) ChemBioChem 9, 826−848.

[2] L Tran, R W Broadhurst, M Tosin, A Cavalli & K J Weissman (2010) Chem. Biol. 17, 705−716.

[3] J Davison, J Dorival, H Rabeharindranto, H Mazon, B Chagot, A Gruez & K J Weissman (2014) Chem. Sci. 5, 3081−3095.

[4] N Pulsawat, S Kitani & T Nihira (2007) Gene 393, 31−42.

[5] J Dorival, T Annaval, F Risser, S Collin, O Bimai, H Lecordier, A Kriznik, C Jacob, A Gruez, B Chagot * K J Weissman, ms in preparation