GNAT-Like Strategy for Polyketide Chain Initiation

Liangcai Gu,^1,2* Todd W. Geders,^1,6* Bo Wang,⁴ William H. Gerwick,⁷ Kristina Håkansson,⁴ Janet L. Smith,^1,3 David H. Sherman^1,2,4,5

An unexpected biochemical strategy for chain initiation is described for the loading module of the polyketide synthase of curacin A, an anticancer lead derived from the marine cyanobacterium Lyngbya majuscula. A central GCN5-related N-acetyltransferase (GNAT) domain bears bifunctional decarboxylase/S-acetyltransferase activity, both unprecedented for the GNAT superfamily. A CurA loading tridomain, consisting of an adaptor domain, the GNAT domain, and an acyl carrier protein, was assessed biochemically, revealing that a domain showing homology to GNAT (GNAT_L) catalyzes (i) decarboxylation of malonyl-coenzyme A (malonyl-CoA) to acetyl-CoA and (ii) direct S-acetyl transfer from acetyl-CoA to load an adjacent acyl carrier protein domain (ACP_L). Moreover, the N-terminal adapter domain was shown to facilitate acetyl-group transfer. Crystal structures of GNAT_L were solved at 1.95 angstroms (ligand-free form) and 2.75 angstroms (acyl-CoA complex), showing distinct substrate tunnels for acyl-CoA and holo-ACP_L binding. Modeling and site-directed mutagenesis experiments demonstrated that histidine-389 and threonine-355, at the convergence of the CoA and ACP tunnels, participate in malonyl-CoA decarboxylation but not in acetyl-group transfer. Decarboxylation precedes acetyl-group transfer, leading to acetyl-ACP_L as the key curacin A starter unit.

¹ Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
² Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
³ Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
⁴ Department of Chemistry, University of Michigan, Ann Arbor, MI48109, USA.
⁵ Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA.
⁶ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
⁷ Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA92093, USA.

^* These authors contributed equally to this work.

To whom correspondence should be addressed. E-mail: davidhs{at}umich.edu (D.H.S.); JanetSmith{at}umich.edu (J.L.S.)

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