Transacylation Kinetics in Fatty Acid and Polyketide Synthases and its Sensitivity to Point Mutations**

Stegemann, Franziska; Grininger, Martin

doi:10.1002/cctc.202002077

Cited by 17 publications

(23 citation statements)

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“…S1). We have recently demonstrated that the promiscuous AT domain from metazoan fatty acid synthase (FAS), termed malonyl-/acetyl transferase (MAT), is able to transfer various acyl-CoA moieties with high efficiencies 13 , different to AT domains from PKSs 14 . We hypothesized that this domain may also transfer fluorinated extender substrates into a PKS module, since FASs and PKSs are structurally and biochemically related (Fig 1b) [15][16][17] .…”

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Directed biosynthesis of fluorinated polyketides

Rittner

Joppe

Schmidt

et al. 2021

Preprint

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Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-specific incorporation of fluorine in complex molecules have improved in recent years, approaches for the direct biosynthetic fluorination of natural compounds are still rare. Herein, we present a broadly applicable approach for site-specific, biocatalytic derivatization of polyketides with fluorine. Specifically, we exchanged the native acyltransferase domain (AT) of a polyketide synthase (PKS), which acts as the gatekeeper for selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase (FAS). The resulting PKS/FAS hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl disubstitutions in polyketide scaffolds. Addition of a fluorine atom is often a decisive factor toward developing superior properties in next-generation antibiotics, including the macrolide solithromycin. We demonstrate the feasibility of our approach in the semisynthesis of a fluorinated derivative of the macrolide antibiotic YC-17.

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Directed biosynthesis of fluorinated polyketides

Rittner

Joppe

Schmidt

et al. 2021

Preprint

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“…Parts of this chapter have been previously accepted for publication in: F. Stegemann, M. Grininger, "Transacylation Kinetics in Fatty Acid and Polyketide Synthases and its Sensitivity to Point Mutations", ChemCatChem (2021). [1] For individual contributions, copyright, and Creative Commons license, please see the statement of personal contributions.…”

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confidence: 99%

“…All elution fractions contained other bands of lower molecular weight proteins. Since purification via tandem a nity chromatography usually results in pure protein, [1,17] these are most likely degraded proteins. The band of 170 kDa could correspond to the protein without KS-AT, the band at 60 kDa to KS-LD, and the band of 45 kDa to KS or LD-AT domains.…”

Section: Chimeric Mfas With Swapped Debs At5 and Raps At14mentioning

confidence: 99%

“…

Teile der vorliegenden Arbeit wurden vorab zur Publikation akzeptiert in: F. Stegemann, M. Grininger, "Transacylation Kinetics in Fatty Acid and Polyketide Synthases and its Sensitivity to Point Mutations", ChemCatChem (2021). [1] Eine detaillierte Auflistung der Inhalte, mein eigener Anteil, Copyright sowie Creative-Commons-Lizenzen sind im Anhang unter dem Abschnitt "Statement of Personal Contributions" dargestellt.In die vorliegende Arbeit sind die Ergebnisse verschiedener Bachelor-und Master-Abschlussarbeiten des FB14 eingegangen, die von mir betreut wurden. Eine detaillierte Auflistung findet sich ebenfalls im Anhang unter dem Abschnitt "Statement of Personal Contributions".

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“…Teile der vorliegenden Arbeit wurden vorab zur Publikation akzeptiert in: F. Stegemann, M. Grininger, "Transacylation Kinetics in Fatty Acid and Polyketide Synthases and its Sensitivity to Point Mutations", ChemCatChem (2021). [1] Eine detaillierte Auflistung der Inhalte, mein eigener Anteil, Copyright sowie Creative-Commons-Lizenzen sind im Anhang unter dem Abschnitt "Statement of Personal Contributions" dargestellt.…”

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Modulating synthetic pathways in megasynthases

Stegemann¹

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Polyketides are highly valuable natural products, which are widely used as pharmaceuticals due to their beneficial characteristics, comprising antibacterial, antifungal, immunosuppressive, and antitumor properties, among others. Their biosynthesis is performed by large and complex multiproteins, the polyketide synthases (PKSs). This study solely focuses on the class of type I PKSs, which arrange all their enzymatic domains on one or more polypeptides. Despite their high medical value, little is known about mechanistic details in PKSs. One central domain is the acyl transferase (AT), which is present in all PKSs and channels small acyl substrates into the enzyme. More precisely, the AT loads the substrates onto the essential acyl carrier protein (ACP), which subsequently shuttles the substrates and all intermediates for condensation and modification to additional domains to build the final polyketide. Some PKSs use their domains several times during biosynthesis and work iteratively – these are called iterative PKSs. Others feature several sets of domains, each being used only once during biosynthesis – these PKSs are called modular PKSs. All PKSs or PKS modules consist of minimum three essential domains to connect the acyl substrates. Three modifying domains are optional and can enlarge the minimal set. According to the domain composition, the acyl substrate is fully reduced, partly reduced, or not reduced at all. This variation of modifying domains accounts for the huge structural and therefore functional variety of polyketides. Even though the structure of fatty acids is not exactly reminiscent of polyketides, their biosynthetic pathways are closely related. Fatty acid biosynthesis is carried out by fatty acid synthases (FASs), which share many similarities with PKSs. Both megasynthases feature the same domains, performing the same reactions to connect and modify small acyl substrates. In contrast to PKSs, FASs always contain one full set of modifying domains which is used iteratively, leading to fully reduced fatty acids. The present thesis extensively analyzes the AT of different PKSs in its substrate selectivity, AT-ACP domain-domain interaction, and enzymatic kinetic properties. The following key findings are revealed through comparison: 1.) ATs of PKSs appear slower than the ones of FASs, which may reflect the different scopes of biosynthetic pathways. Fatty acids as essential compounds in all organisms are needed in high amounts for physiological functions, whereas polyketides as secondary metabolites only require basal concentrations to take effect. 2.) The slower ATs from modular PKSs do not load non-native substrates even in absence of the native substrates. This is different to the faster ATs from iterative PKSs and FASs, which indicates high substrate specificity solely for the ATs from modular PKSs and emphasizes their role as gatekeepers in polyketide synthesis. 3.) The substrate selectivity can emerge in either the first or the second step of the AT-mediated ACP loading and is not assured by a hydrolytic proofreading function. Moreover, a mutational study on the AT-ACP interaction in the modular PKS 6-deoxyerythronolide B synthase (DEBS) shows that single surface point mutations can influence AT-mediated reactions in a complex manner. Data reveals high enzyme kinetic plasticity of the AT-ACP interaction, which was also recently demonstrated for the interaction in a type II FAS. Based on these findings, the mammalian FAS is engineered towards a modular PKS-like as- sembly line with the long-term goal to rationally synthesize new products. Basically, three important aspects need to be considered: 1.) AT’s loading needs to be splitted in specific loading of a priming substrate by a priming AT and in specific loading of an elongation substrate by an elongation AT. 2.) FAS-based elongation modules need to be designed with varying domain compositions for introducing functional groups in the product. 3.) Covalent and non-covalent linkers need to be designed for connection of priming and elongation modules. This study focuses on the first aspect, splitting loading of priming and elongation substrates. An elongation substrate-specific AT is installed in the mammalian FAS via domain swapping. Since ATs from modular PKSs were proven to be substrate specific, these are used to exchange the mammalian FAS AT. This work demonstrates that it is extremely challenging to create stable and functional chimeras, but first essential steps are taken. Proper domain boundaries for AT swapping are established and a stable chimera with 70 % wild type AT activity is created. However, this chimera is only of limited value for application in an elongation module due to the intrinsic slow turnover rate of the wild type AT. Using another PKS AT, a stable elongation module is designed and analyzed in its activity in combination with a priming module. These experiments demonstrate that the loading of priming substrates are successfully suppressed in the elongation module, but nonetheless only minor turnover rates are detected in the assembly line. ...

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Wie Acyl‐Carrier‐Proteine (ACPs) die Biosynthese von Fettsäuren und Polyketiden steuern

Buyachuihan,

Stegemann,

Grininger

2023

Angewandte Chemie

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Megasynthases, such as type I fatty acid and polyketide synthases (FASs and PKSs), are multienzyme complexes responsible for producing primary metabolites and complex natural products. Fatty acids (FAs) and polyketides (PKs) are built by assembling and modifying small acyl moieties in a stepwise manner. A central aspect of FA and PK biosynthesis involves the shuttling of substrates between the domains of the multienzyme complex. This essential process is mediated by small acyl carrier proteins (ACPs). The ACPs must navigate to the different catalytic domains within the multienzyme complex in a particular order to guarantee the fidelity of the biosynthesis pathway. However, the precise mechanisms underlying ACP‐mediated substrate shuttling, particularly the factors contributing to the programming of the ACP movement, still need to be fully understood. This review illustrates the current understanding of substrate shuttling, including concepts of conformational and specificity control, and proposes a confined ACP movement within type I megasynthases.

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Transacylation Kinetics in Fatty Acid and Polyketide Synthases and its Sensitivity to Point Mutations**

Cited by 17 publications

References 50 publications

Directed biosynthesis of fluorinated polyketides

Directed biosynthesis of fluorinated polyketides

Modulating synthetic pathways in megasynthases

Wie Acyl‐Carrier‐Proteine (ACPs) die Biosynthese von Fettsäuren und Polyketiden steuern

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