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Mutagenesis supports AlphaFold prediction of how modular polyketide synthase acyl carrier proteins dock with downstream ketosynthases
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  • Adrian Keatinge-Clay,
  • Melissa Hirsch,
  • Ronak R. Desai,
  • Shreyas Annaswamy
Adrian Keatinge-Clay
The University of Texas at Austin Department of Molecular Biosciences

Corresponding Author:[email protected]

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Melissa Hirsch
The University of Texas at Austin Department of Chemistry
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Ronak R. Desai
The University of Texas at Austin Department of Molecular Biosciences
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Shreyas Annaswamy
The University of Texas at Austin Department of Molecular Biosciences
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Abstract

The docking of an acyl carrier protein (ACP) domain with a downstream ketosynthase (KS) domain in each module of a polyketide synthase (PKS) helps ensure accurate biosynthesis. If the polyketide chain bound to the ACP has been properly modified by upstream processing enzymes and is compatible with gatekeeping residues in the KS tunnel, a transacylation reaction can transfer it from the 18.1-Å phosphopanthetheinyl arm of the ACP to the reactive cysteine of the KS. AlphaFold-Multimer predicts a general interface for these transacylation checkpoints. Half of the solutions obtained for 50 ACP/KS pairs show the KS motif TxLGDP forming the first turn of an a-helix, as in reported structures, while half show it forming a type I b-turn not previously observed. Solutions with the latter conformation may represent how these domains are relatively positioned during the transacylation reaction, as the entrance to the KS active site is relatively open and the phosphopantetheinylated ACP serine and the reactive KS cysteine are relatively closer - 17.2 Å vs. 20.9 Å, on average. To probe the predicted interface, 20 mutations were made to KS surface residues within the model triketide lactone synthase P1- P6- P7. The activities of these mutants are consistent with the proposed interface.
10 Feb 2024Submitted to PROTEINS: Structure, Function, and Bioinformatics
15 Feb 2024Submission Checks Completed
15 Feb 2024Assigned to Editor
11 Apr 2024Review(s) Completed, Editorial Evaluation Pending
15 May 2024Editorial Decision: Revise Minor
05 Jun 20241st Revision Received
13 Jun 2024Submission Checks Completed
13 Jun 2024Assigned to Editor
13 Jun 2024Review(s) Completed, Editorial Evaluation Pending
13 Jun 2024Reviewer(s) Assigned