title: Protein-protein recognition between acyltransferases and acyl carrier proteins in multimodular polyketide synthases.
abstract: Acyltransferase (AT) domains of multimodular polyketide synthases are the primary gatekeepers for stepwise incorporation of building blocks into a growing polyketide chain. Each AT domain has two substrates, an alpha-carboxylated CoA thioester (e.g., malonyl-CoA or methylmalonyl-CoA) and an acyl carrier protein (ACP). Whereas the acyl-CoA specificity of AT domains has been extensively investigated, little is known about their ACP specificity. Guided by recent high-resolution structural insights, we have systematically probed the protein-protein interactions between AT domains, ACP domains, and the linkers that flank AT domains. Representative AT domains of the 6-deoxyerythronolide B synthase (DEBS) have greater than 10-fold specificity for their cognate ACP substrates as compared to other ACP domains from the same synthase. Both of the flanking (N- and C-terminal) linkers of an AT domain contributed to the efficiency and specificity of transacylation. As a frame of reference, the activity and specificity of a stand-alone AT domain from the "AT-less" disorazole synthase (DSZS) were also quantified. The activity (k(cat)/K(M)) of this AT was >250-fold higher than the corresponding values for DEBS AT domains. Although the AT from DSZS discriminated modestly against ACP domains from DEBS, it exhibited >40-fold higher activity in trans in the presence of these heterologous substrates than their natural AT domains. Our results highlight the opportunity for regioselective modification of a polyketide backbone by in trans complementation of inactivated AT domains. They also reinforce the need for more careful consideration of protein-protein interactions in the engineering of these assembly line enzymes.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
authors.forename: Fong T
authors.initials: FT
authors.lastname: Wong
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Chen
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000378:metabolism*; D000215:Acylation; D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D048630:Polyketide Synthases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D000213:Acyl Carrier Protein; D048630:Polyketide Synthases; D000217:Acyltransferases