Structural studies of components from the minimal polyketide and fatty acid synthases from Streptomyces coelicolor

Abstract

Polyketides are small biologically active molecules synthesised by large enzyme complexes, termed pdlyketide synthases (PKS), in response to environmental factors. PKS's operate in a similar repetitive way to fatty acid synthases (F AS). The initial stages of both processes rely on similar enzymes, some of which have been characterised structurally by NMR or x-ray crystallography. However, to date, the structures and interactions in both systems have not been compared. This work looks at the central role played by the acyl carrier proteins (ACP) ofPKS and FAS systems in Streptomyces coelicolor, and the role played by the ketosynthase/chain length factor (KS/CLF) from the same species. The PKS ACP structure has been published previously allowing comparison with the F AS ACP structure studied here. Samples of unlabelled, lSN and lSN/ l3C FAS ACP from Streptomyces coelicolor were expressed in E. coli and purified using anionic exchange chromatography. A range of 2D and 3D double and triple resonance NMR spectra were recorded and analysed in order to use an automated assignment program to assign chemical shift resonances for nitrogen eSN), carbon (l3C) and hydrogen eH) atoms within the protein. NOE data was used to generate a list of distance restraints, which were fed into a simulated annealing protocol to generate an ensemble of three dimensional structures for FAS ACP. Structural analysis has shown that, although the F AS and PKS ACPs from S. coelicolor share the same overall tertiary structure, subtle differences are observed. The orientation of helix I differs by ~20° between the two structures whilst the loop region between helices I and II is highly flexible in PKS ACP, but is well ordered within the FAS ACP structure. The gris PKS ACP from Streptomyces griseus has been crystallised in conditions containing ammonium sulphate, sodium chloride and tris buffer, and two data sets collected in house at 2.5 A and 1.8 A. Data has been processed using the CCP4 suite of programs. Molecular replacement, using a variety of different ACP models, has so far proved unsuccessful. Whilst heavy atom soaks using gadolinium and samarium have failed to produce a heavy atom derivative. Attempts have been made to crystallise a sample of KS/CLF complex from Streptomyces coelicolor, provided by the University of Bristol, using standard methods. This complex is, however, very unstable and precipitates, with a loss of activity, between minutes to hours. Therefore attempts to crystallise the complex with the act ACP are to be attempted in the hope that this will help to stabilise the complex.

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