Abstract

Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthases: Versatility from a unique substrate channel.

Tsai, S.-C., Miercke, L.J.W., Krucinski, J., Gokhale, R., Chen, Julian C.-H., Foster, P.G., Cane, D.E., Khosla, C., and Stroud, R.M.

PNAS 98 26 14808-14813. (2001)
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As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous diffraction and refined to an R factor of 23.1% to 2.8? resolution. Its overall tertiary architecture belongs to the alpha/beta-hydrolase family, with two unusal features unprecedented in this family: a hydrophobic, leucine-rich dimer interface and a substrate channel that passes throgh the entire protein. The active site triad, comprised of Asp169, His259 and Ser142, is located in the middle of the substrate channel, suggesting passage of the substrate through the protein.

Modeling indicated that the active site can accomidate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explains the observed substrate specificity of this TE and offers strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the two-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangment of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular PKSs have a similar fold, dimer two-fold axis, and a substrate channel geometry.

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