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ABSTRACT Polyketide synthases (PKSs) are multi-domain enzymatic assembly lines that biosynthesise a wide selection of bioactive natural products from simple building blocks. In contrast to theircis-acyltransferase (AT) counterparts,trans-AT PKSs rely on stand-alone AT domains to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery.Trans-AT PKS gene clusters also encode acyl hydrolase (AH) domains, which are predicted to share the overall fold of AT domains, but hydrolyse aberrant acyl chains from ACP domains, thus ensuring efficient polyketide biosynthesis. How such domains specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH domain and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AH and ACP domains contributes to recognition of cognate and non-cognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the production of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis bytrans-AT PKSs, and provide foundations for future bioengineering efforts.
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Exploring the compatibility of phosphopantetheinyl transferases with acyl carrier proteins spanning type II polyketide synthase sequence space
Abstract Phosphopantetheinyl transferases (PPTases) play an essential role in primary and secondary metabolism. These enzymes facilitate the post-translational activation of acyl carrier proteins (ACPs) central to the biosynthesis of fatty acids and polyketides. Modulation of ACP-PPTase interactions is a promising approach to both increase access to desired molecular outputs and disrupt mechanisms associated with disease progression. However, such an approach requires understanding the molecular principles that govern ACP-PPTase interactions across diverse synthases. Through a multi-year, course-based undergraduate research experience (CURE), 17 ACPs representing a range of putative type II polyketide synthases, from actinobacterial and non-actinobacterial phyla, were evaluated as substrates for three PPTases (AcpS, Sfp, and vulPPT). The observed PPTase compatibility, sequence-level analyses, and predictive structural modelling suggest that ACP selectivity is driven by amino acids surrounding the conserved, modified serine on the ACP. We propose that vulPPT and Sfp are driven primarily by hydrophobic contacts, whereas AcpS may favor ACPs which exhibit high net-negative charge density, as well as a broad electronegative surface distribution. Furthermore, we report a plausible, hitherto unreported hydrophobic interaction between vulPPT and a conserved ACP crease, upstream of the invariant serine, which may facilitate docking. This work provides a catalog of compatible and incompatible ACP-PPTase partnerships, highlighting specific regions on the ACP and/or PPTase that show promise for future strategic engineering and inhibitor development efforts.
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- Award ID(s):
- 2201984
- PAR ID:
- 10644136
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Journal of Industrial Microbiology and Biotechnology
- ISSN:
- 1367-5435
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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