Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase.
Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ-containing loading modules are shown here to contain a key arginine residue at their active site. Site-specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13-methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex-tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1-TE, in which the 4'-phosphopante-theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro-ducts in detectable amounts.
Long
Paul F
PF
Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
Wilkinson
Christopher J
CJ
Bisang
Christian P
CP
Cortés
Jesús
J
Dunster
Nicholas
N
Oliynyk
Marko
M
McCormick
Ellen
E
McArthur
Hamish
H
Mendez
Carmen
C
Salas
José A
JA
Staunton
James
J
Leadlay
Peter F
PF
D000217:Acyltransferases / Q000737:chemistry; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D004917:Erythromycin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009827:Oleandomycin / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D015202:Protein Engineering; D016930:Saccharopolyspora / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D009097:Multienzyme Complexes; D004917:Erythromycin; D000217:Acyltransferases; D009827:Oleandomycin