{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T12:13:38Z","timestamp":1790252018480,"version":"4.1.0"},"reference-count":30,"publisher":"American Chemical Society (ACS)","issue":"23","license":[{"start":{"date-parts":[[2015,5,28]],"date-time":"2015-05-28T00:00:00Z","timestamp":1432771200000},"content-version":"vor","delay-in-days":365,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["K12-GM088033"],"award-info":[{"award-number":["K12-GM088033"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 GM022172"],"award-info":[{"award-number":["R01 GM022172"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 GM087934"],"award-info":[{"award-number":["R01 GM087934"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2014,6,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>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 &amp;gt;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 (kcat\/KM) 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.<\/jats:p>","DOI":"10.1021\/bi5004316","type":"journal-article","created":{"date-parts":[[2014,5,28]],"date-time":"2014-05-28T12:28:16Z","timestamp":1401280096000},"page":"3796-3806","source":"Crossref","is-referenced-by-count":55,"title":["Comparative Analysis of the Substrate Specificity\nof\n                    <i>trans<\/i>\n                    - versus\n                    <i>cis-<\/i>\n                    Acyltransferases\nof Assembly Line Polyketide Synthases"],"prefix":"10.1021","volume":"53","author":[{"given":"Briana J.","family":"Dunn","sequence":"first","affiliation":[{"name":"Stanford University , , , ,","place":["Stanford, California, United States, 94305"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharine R.","family":"Watts","sequence":"additional","affiliation":[{"name":"Stanford University , , , ,","place":["Stanford, California, United States, 94305"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Robbins","sequence":"additional","affiliation":[{"name":"Stanford University , , , ,","place":["Stanford, California, United States, 94305"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David E.","family":"Cane","sequence":"additional","affiliation":[{"name":"Brown University , , Box H, , ,","place":["Providence, Rhode Island, United States, 02912-9108"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chaitan","family":"Khosla","sequence":"additional","affiliation":[{"name":"Stanford University , , , ,","place":["Stanford, California, United States, 94305"]},{"name":"Stanford University , , , ,","place":["Stanford, California, United States, 94305"]},{"name":"Stanford University , , , ,","place":["Stanford, California, United States, 94305"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2014,5,28]]},"reference":[{"key":"2026090120152043400_cit1","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1021\/ar7000414","article-title":"The chemical\nversatility of natural-product assembly lines","volume":"41","author":"Walsh","year":"2008","journal-title":"Acc. Chem. Res."},{"key":"2026090120152043400_cit2","doi-asserted-by":"crossref","DOI":"10.1098\/rsif.2013.0297","article-title":"Engineering\nthe acyltransferase substrate specificity\nof assembly line polyketide synthases","volume":"10","author":"Dunn","year":"2013","journal-title":"J. R.\nSoc. Interface"},{"key":"2026090120152043400_cit3","doi-asserted-by":"crossref","first-page":"996","DOI":"10.1039\/b816430b","article-title":"Biosynthesis\nof polyketides by trans-AT polyketide synthases","volume":"27","author":"Piel","year":"2010","journal-title":"Nat. Prod. Rep."},{"key":"2026090120152043400_cit4","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.gene.2005.06.003","article-title":"The biosynthetic\ngenes for disorazoles, potent cytotoxic compounds that disrupt microtubule\nformation","volume":"359","author":"Carvalho","year":"2005","journal-title":"Gene"},{"key":"2026090120152043400_cit5","doi-asserted-by":"crossref","first-page":"3149","DOI":"10.1073\/pnas.0537286100","article-title":"Type I polyketide synthase requiring\na discrete acyltransferase for polyketide biosynthesis","volume":"100","author":"Cheng","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U S A"},{"key":"2026090120152043400_cit6","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1016\/j.chembiol.2008.09.013","article-title":"In vivo\nand in vitro trans-acylation by BryP, the putative bryostatin pathway\nacyltransferase derived from an uncultured marine symbiont","volume":"15","author":"Lopanik","year":"2008","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit7","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1016\/j.chembiol.2011.02.007","article-title":"Supramolecular templating\nin kirromycin biosynthesis: the acyltransferase KirCII loads ethylmalonyl-CoA\nextender onto a specific ACP of the trans-AT PKS","volume":"18","author":"Musiol","year":"2011","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit8","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1021\/cb3003489","article-title":"Poly specific trans-acyltransferase\nmachinery revealed via engineered acyl-CoA synthetases","volume":"8","author":"Koryakina","year":"2013","journal-title":"ACS Chem. Biol."},{"key":"2026090120152043400_cit9","doi-asserted-by":"crossref","first-page":"14307","DOI":"10.1021\/ja037429l","article-title":"Enhancing the modularity\nof the modular polyketide synthases: transacylation in modular polyketide\nsynthases catalyzed by malonyl-CoA:ACP transacylase","volume":"125","author":"Kumar","year":"2003","journal-title":"J. Am. Chem. Soc."},{"key":"2026090120152043400_cit10","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1021\/bi901826g","article-title":"Protein\u2013protein\nrecognition between acyltransferases and acyl carrier proteins in\nmultimodular polyketide synthases","volume":"49","author":"Wong","year":"2010","journal-title":"Biochemistry"},{"key":"2026090120152043400_cit11","doi-asserted-by":"crossref","first-page":"1839","DOI":"10.1021\/bi400185v","article-title":"Mechanism\nand specificity of an acyltransferase\ndomain from a modular polyketide synthase","volume":"52","author":"Dunn","year":"2013","journal-title":"Biochemistry"},{"key":"2026090120152043400_cit12","doi-asserted-by":"crossref","first-page":"16809","DOI":"10.1021\/ja409048k","article-title":"In vitro reconstitution\nand analysis\nof the 6-deoxyerythronolide B synthase","volume":"135","author":"Lowry","year":"2013","journal-title":"J. Am.\nChem. Soc."},{"key":"2026090120152043400_cit13","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1016\/j.chembiol.2007.05.015","article-title":"Structure-based\ndissociation of a\ntype I polyketide synthase module","volume":"14","author":"Chen","year":"2007","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit14","doi-asserted-by":"crossref","first-page":"13892","DOI":"10.1021\/bi048418n","article-title":"Reconstituting\nmodular activity from separated domains\nof 6-deoxyerythronolide B synthase","volume":"43","author":"Kim","year":"2004","journal-title":"Biochemistry"},{"key":"2026090120152043400_cit15","doi-asserted-by":"crossref","first-page":"6539","DOI":"10.1021\/bi200632j","article-title":"Structure and mechanism\nof the trans-acting acyltransferase from the disorazole synthase","volume":"50","author":"Wong","year":"2011","journal-title":"Biochemistry"},{"key":"2026090120152043400_cit16","doi-asserted-by":"crossref","first-page":"1790","DOI":"10.1126\/science.1058092","article-title":"Biosynthesis\nof complex polyketides in a metabolically engineered strain of E.\ncoli","volume":"291","author":"Pfeifer","year":"2001","journal-title":"Science"},{"key":"2026090120152043400_cit17","doi-asserted-by":"crossref","first-page":"31630","DOI":"10.1016\/S0021-9258(18)31741-1","article-title":"Inhibition\nof the human methylmalonyl-CoA mutase by various CoA-esters","volume":"269","author":"Taoka","year":"1994","journal-title":"J. Biol. Chem."},{"key":"2026090120152043400_cit18","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.chembiol.2007.12.009","article-title":"Molecular\nanalysis of the kirromycin biosynthetic gene cluster revealed beta-alanine\nas precursor of the pyridone moiety","volume":"15","author":"Weber","year":"2008","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit19","doi-asserted-by":"crossref","first-page":"1277","DOI":"10.1002\/cbic.200400459","article-title":"Production of the tubulin\ndestabilizer disorazol in Sorangium cellulosum: Biosynthetic machinery and regulatory genes","volume":"6","author":"Kopp","year":"2005","journal-title":"ChemBioChem"},{"key":"2026090120152043400_cit20","doi-asserted-by":"crossref","first-page":"931","DOI":"10.1016\/j.chembiol.2007.07.012","article-title":"Structural\nand mechanistic analysis of protein interactions in module 3 of the\n6-deoxyerythronolide B synthase","volume":"14","author":"Tang","year":"2007","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit21","doi-asserted-by":"crossref","first-page":"636","DOI":"10.1016\/j.chembiol.2014.02.019","article-title":"Reprogramming Acyl\nCarrier Protein Interactions of an Acyl-CoA Promiscuous trans-Acyltransferase","volume":"21","author":"Ye","year":"2014","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit22","doi-asserted-by":"crossref","first-page":"1343","DOI":"10.1002\/cbic.201300211","article-title":"The AT(2) domain of KirCI loads malonyl extender units to the ACPs\nof the kirromycin PKS","volume":"14","author":"Musiol","year":"2013","journal-title":"ChemBioChem"},{"key":"2026090120152043400_cit23","doi-asserted-by":"crossref","first-page":"2093","DOI":"10.1110\/ps.073011407","article-title":"Solution structure\nand proposed domain domain recognition interface of an acyl carrier\nprotein domain from a modular polyketide synthase","volume":"16","author":"Alekseyev","year":"2007","journal-title":"Protein Sci."},{"key":"2026090120152043400_cit24","doi-asserted-by":"crossref","first-page":"1075","DOI":"10.1016\/j.chembiol.2011.07.016","article-title":"Acyl-CoA subunit\nselectivity in the pikromycin polyketide synthase PikAIV: Steady-state\nkinetics and active-site occupancy analysis by FTICR-MS","volume":"18","author":"Bonnett","year":"2011","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit25","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.chembiol.2012.01.005","article-title":"Polyketide\nproofreading by an acyltransferase-like enzyme","volume":"19","author":"Jensen","year":"2012","journal-title":"Chem. Biol."},{"key":"2026090120152043400_cit26","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1146\/annurev.biochem.76.053105.093515","article-title":"Structure\nand mechanism of the 6-deoxyerythronolide B synthase","volume":"76","author":"Khosla","year":"2007","journal-title":"Annu. Rev. Biochem."},{"key":"2026090120152043400_cit27","doi-asserted-by":"crossref","first-page":"2524","DOI":"10.1021\/bi971887n","article-title":"Functional orientation of the acyltransferase\ndomain in a module of the erythromycin polyketide synthase","volume":"37","author":"Gokhale","year":"1998","journal-title":"Biochemistry"},{"key":"2026090120152043400_cit28","first-page":"75","article-title":"The utilization\nof binding energy in coupled vectorial processes","volume":"51","author":"Jencks","year":"1980","journal-title":"Adv. Enzymol. Relat. Areas Mol. Biol."},{"key":"2026090120152043400_cit29","doi-asserted-by":"crossref","first-page":"1136","DOI":"10.1002\/jms.1240","article-title":"Analysis\nof intracellular short organic acid-coenzyme A esters from actinomycetes\nusing liquid chromatography-electrospray ionization-mass spectrometry","volume":"42","author":"Park","year":"2007","journal-title":"J. Mass Spectrom."},{"key":"2026090120152043400_cit30","doi-asserted-by":"crossref","first-page":"3701","DOI":"10.1007\/s00253-013-5503-8","article-title":"Characterization\nand engineering of the ethylmalonyl-CoA pathway towards the improved\nheterologous production of polyketides in Streptomyces venezuelae","volume":"98","author":"Jung","year":"2014","journal-title":"Appl. Microbiol.\nBiotechnol."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/bi5004316","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/23\/3796\/7890328\/bi5004316.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/23\/3796\/7890328\/bi5004316.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T00:15:31Z","timestamp":1788308131000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/53\/23\/3796\/774233\/Comparative-Analysis-of-the-Substrate-Specificity"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,5,28]]},"references-count":30,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2014,5,28]]},"published-print":{"date-parts":[[2014,6,17]]}},"URL":"https:\/\/doi.org\/10.1021\/bi5004316","relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.718424189.793514535","asserted-by":"object"}]},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,5,28]]}}}