title: GNAT-like strategy for polyketide chain initiation.
abstract: 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.
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Liangcai
authors.initials: L
authors.lastname: Gu
authors.forename: Todd W
authors.initials: TW
authors.lastname: Geders
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.forename: William H
authors.initials: WH
authors.lastname: Gerwick
authors.forename: Kristina
authors.initials: K
authors.lastname: Håkansson
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism*; D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D003521:Cyclopropanes / Q000378:metabolism*; D003653:Decarboxylation; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013844:Thiazoles / Q000378:metabolism*
chemical_list: D000213:Acyl Carrier Protein; D003521:Cyclopropanes; D013844:Thiazoles; C095843:curacin A; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D048630:Polyketide Synthases; D000123:Acetyltransferases; D002262:Carboxy-Lyases