title: Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases.
abstract: Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (k(cat)/K(M)) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.
authors.affiliation: Department of Chemical Engineering, ‡Department of Chemistry, and ∥Department of Biochemistry, Stanford University , Stanford, California 94305, United States.
authors.forename: Briana J
authors.initials: BJ
authors.lastname: Dunn
authors.forename: Katharine R
authors.initials: KR
authors.lastname: Watts
authors.forename: Thomas
authors.initials: T
authors.lastname: Robbins
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D018942:Macrolides / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010080:Oxazoles / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061065:Polyketides / Q000378:metabolism; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011728:Pyridones / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D018942:Macrolides; D050505:Mutant Proteins; D010080:Oxazoles; D010446:Peptide Fragments; D061065:Polyketides; D021122:Protein Subunits; D011728:Pyridones; D011993:Recombinant Fusion Proteins; C102829:ethylmalonyl-coenzyme A; C015357:methylmalonyl-coenzyme A; D008316:Malonyl Coenzyme A; D048630:Polyketide Synthases; D000217:Acyltransferases; C008536:mocimycin