Fatty acid and polyketide synthases (FASs and PKSs) synthesize physiologically and pharmaceutically important products by condensation of acyl building blocks. The transacylation reaction catalyzed by acyl transferases (ATs) is responsible for the selection of acyl‐CoA esters for further processing by FASs and PKSs. In this study, the AT domains of different multidomain (type I) PKS systems are kinetically described in their substrate selectivity, AT−Acyl carrier protein (ACP) domain‐domain interaction and enzymatic kinetic properties. We observe that the ATs of modular PKSs, intricate protein complexes occurring in bacteria and responsible for the biosynthesis of bioactive polyketides, are significantly slower than ATs of mammalian FASs, reflecting the respective purpose of the biosynthetic pathways within the organism and their metabolic context. We further perform a mutational study on the kinetics of the AT−ACP interaction in the modular PKS 6‐deoxyerythronolide B synthase (DEBS) and find a high plasticity in enzyme properties, which we explain by a high plasticity in AT−ACP recognition. Our study enlarges the understanding of ATs in its molecular properties and is similarly a call for thorough AT‐centered PKS engineering strategies.

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. ...

The Front Cover shows an example of polyketide synthases, producing complex products from simple substrates, in which the acyl transferase (AT) selects substrates from the bulk cytoplasm. Subsequently, the substrates are transferred by the acyl carrier protein (ACP) robotic arm to the catalytic domains KS, KR, DH, ER forcondensation and processing. Secondary metabolite biosynthetic pathways often proceed separately from the housekeeping functions of a cell. Compartmentalization is achieved by enzyme kinetic phenomena that may rely on dedicated protein architecture and physical proximity. The substrates and cofactors for secondary metabolite biosynthetic pathways are at least partly shared with primary metabolism, and questions remain how resources are distributed among primary and secondary metabolisms. In their Full Paper, F. Stegemann and M. Grininger analyze the process of substrate loading in enzyme kinetic detail, in its substrate selectivity and in its protein‐protein interaction. More information can be found in the Full Paper by F. Stegemann and M. Grininger.

Fatty acid and polyketide synthases (FASs and PKSs) synthesize physiologically and pharmaceutically important products by condensation of acyl building blocks. In both multidomain enzymes, the acyl transferase (AT) is the key player responsible for the selection of these acyl units for further processing. In this study, the AT domains of different PKS systems are kinetically described in their substrate selectivity, AT–Acyl carrier protein (ACP) domain-domain interaction, and enzymatic kinetic properties. The ATs of modular PKSs, the proteins assembling the most intricate polyketides, turned out to be significantly slower than ATs from iterative FAS and PKS systems, but also more substrate specific. We explain these substantially different properties by the phylogenetically early splitting of species. For the AT of the 6-deoxyerythronolide B synthase (DEBS), the interaction with ACP is analyzed in detail by site-directed mutagenesis of interface residues. Among others, a surface exposed arginine (R850) was replaced by three different residues, leading to mutants with severely different kinetics that cannot be explained by simple effects. Our study enlarges the understanding of ATs in its molecular properties, and is similarly a call for thorough AT-centered PKS engineering strategies.

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