GNAT-like strategy for polyketide chain initiation.
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.
Gu
Liangcai
L
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
Geders
Todd W
TW
Wang
Bo
B
Gerwick
William H
WH
Håkansson
Kristina
K
Smith
Janet L
JL
Sherman
David H
DH
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*
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