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Structural basis for the formation of acylalkylpyrones from two β-ketoacyl units by the fungal type III polyketide synthase CsyB.

Research paper by Takahiro T Mori, Dengfeng D Yang, Takashi T Matsui, Makoto M Hashimoto, Hiroyuki H Morita, Isao I Fujii, Ikuro I Abe

Indexed on: 08 Jan '15Published on: 08 Jan '15Published in: Journal of Biological Chemistry



Abstract

The acylalkylpyrone synthase CsyB from Aspergillus oryzae catalyzes the one-pot formation of the 3-acyl-4-hydroxy-6-alkyl-α-pyrone scaffold from acetoacetyl-CoA, fatty acyl-CoA, and malonyl-CoA. This is the first type III polyketide synthase that performs not only the polyketide chain elongation but also the condensation of two β-ketoacyl units. The crystal structures of wild-type CsyB and its I375F and I375W mutants were solved at 1.7-, 2.3-, and 2.0-Å resolutions, respectively. The crystal structures revealed a unique active site architecture featuring a hitherto unidentified novel pocket for accommodation of the acetoacetyl-CoA starter in addition to the conventional elongation/cyclization pocket with the Cys-His-Asn catalytic triad and the long hydrophobic tunnel for binding the fatty acyl chain. The structures also indicated the presence of a putative nucleophilic water molecule activated by the hydrogen bond networks with His-377 and Cys-155 at the active site center. Furthermore, an in vitro enzyme reaction confirmed that the (18)O atom of the H2(18)O molecule is enzymatically incorporated into the final product. These observations suggested that the enzyme reaction is initiated by the loading of acetoacetyl-CoA onto Cys-155, and subsequent thioester bond cleavage by the nucleophilic water generates the β-keto acid intermediate, which is placed within the novel pocket. The second β-ketoacyl unit is then produced by polyketide chain elongation of fatty acyl-CoA with one molecule of malonyl-CoA, and the condensation with the β-keto acid generates the final products. Indeed, steric modulation of the novel pocket by the structure-based I375F and I375W mutations resulted in altered specificities for the chain lengths of the substrates.