Biology subjects

Grininger, M.

Publications and source records attributed to Grininger, M..

17 recordsLinked to original sources

bioRxiv2025

Switching Metazoan Fatty Acid Synthase Between Reducing and Non-reducing Elongation Mode via Programming of the Ketoreductase Domain

Polyketides constitute a large class of natural products with important biological activities and applications such as antibiotics, antitumor agents, pesticides, and pigments. Their biosynthesis is catalyzed by polyketide synthases (PKSs) which are multi-domain enzymes evolutionarily related to fatty acid synthases (FASs). Despite their close homology in structure and the chemistry they perform, FASs and PKSs differ fundamentally in their catalytic programming: FASs run fully reducing elongation reactions to yield saturated fatty acids, while iterative PKSs execute reductions just in selected cycles, generating complex oxidized compounds. In this study, we aimed at engineering the metazoan FAS in its KR domain to switch from fully reducing to a non-reducing mode during chain elongation. Guided by recent insights into KR programming, we incorporated a helix into metazoan FAS, which is found in KRs from iterative PKSs and type II FASs with chain length programming. These FAS variants initially catalyze complete fatty acid cycles but lose the ability of {beta}-keto reduction in later elongation rounds, producing intermediates that spontaneously cyclize to pyrone products. Finally, our study provides valuable insight into the mechanism of KR catalysis identifying another amino acid next to the active tyrosine which is capable for intermediate protonation.

biochemistry↗

bioRxiv2025

Identification of pseudotetraivprolide from Pseudomonas entomophila give novel insights into the biosynthesis of detoxin/rimosamide-like anti-antibiotics

Novel variants of known natural product (NP) classes might guide our understanding of biosynthesis, mode of action and potential application as drugs for all members of such NP classes. Here we describe a novel member of the widespread detoxine/rimosamide-like (DRL) natural products named pseudotetraivprolide from Pseudomonas strains, which has the characteristic DRL-activity of protecting Bacillus cereus against the antibiotic blasticidin S. The generation of several deletion and complemented mutants, heterologous expression experiments, identification and structure elucidation of several derivatives, chemical synthesis of main derivatives, enzymatic characterization of individual biochemical steps and detailed homology modelling of enzyme complexes were performed. This allowed us to show the primary metabolism-derived malonyl CoA-ACP transacylase (AT) FabD acting as trans-AT in the biosynthesis, suggest an order for all late-stage modifications and provide a function for the three conserved hypothetical proteins PipDFG acting as last-step acetylation complex thereby stabilizing the final product.

microbiology↗

bioRxiv2024

The Kinetics of Carbon-Carbon-Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the {beta}-ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon-carbon-bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here we report enzyme kinetics of the KS-mediated carbon-carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS-mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. Intriguingly, the KS protomers within the dimer exhibit positive cooperativity, investigated by mutational studies and acyl-carrier analysis, which likely serves the regulation of biosynthesis. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

biochemistry↗

bioRxiv2023

Docking Domain Engineering in a Modular Polyketide Synthase and its Impact on Structure and Function

Modular polyketide synthases (PKSs) are attractive targets for the directed, biosynthetic production of platform chemicals and pharmaceuticals by protein engineering. In this study, we analyze docking domains from the 6-deoxyerythronolide B synthase, SYNZIP domains, and the SpyCatcher:SpyTag complex as engineering tools to couple the polypeptides VemG and VemH to functional venemycin synthases. Our data show that the high-affinity interaction or covalent connection of modules, enabled by SYNZIP domains and the SpyCatcher:SpyTag complex, can be advantageous, e.g., in synthesis at low protein concentrations, but their rigidity and steric demand decrease synthesis rates. However, we also show that efficiency can be recovered when inserting a hinge region distant from the rigid interface. This study demonstrates that engineering approaches should take the conformational properties of modular PKSs into account and establishes a three-polypeptide split-venemycin synthase as an exquisite in vitro platform for the analysis and engineering of modular PKSs.

biochemistry↗

bioRxiv2021

Directed biosynthesis of fluorinated polyketides

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.

biochemistry↗

bioRxiv2020

Comparative Study on Acyl Transferases in Fatty Acid and Polyketide Synthases

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.

biochemistry↗

bioRxiv2020

Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding and partly impossible owing to the lack of specific functions or substrates in the host cell, its sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we demonstrate that cell-free systems can be useful in driving the characterization and engineering of biosynthetic pathways. We show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. We prove the concept of this approach on the direct synthesis of indigoidine and rhabdopeptides with the in vitro produced multidomain megasynthases BpsA and KJ12ABC. The in vitro produced proteins are analyzed in detail, i.e., in yield, quality, post-translational modification and specific activity, and compared to recombinantly produced proteins. Our study highlights cell-free PURE systems as suitable setting for the rapid engineering of biosynthetic pathways.

bioengineering↗

bioRxiv2020

Dodecin as carrier protein for immunizations and bioengineering applications

In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signaling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDods ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP). For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/990861v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1b30641org.highwire.dtl.DTLVardef@5fa8bdorg.highwire.dtl.DTLVardef@3f16a3org.highwire.dtl.DTLVardef@1e756a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

Modular polyketide synthases (PKSs) produce complex, bioactive secondary metabolites in assembly line-like multistep reactions. Longstanding efforts to produce novel, biologically active compounds by recombining intact modules to new modular PKSs have mostly resulted in poorly active chimeras and decreased product yields. Recent findings demonstrate that the low efficiencies of modular chimeric PKSs also result from rate limitations in the transfer of the growing polyketide chain across the non-cognate module:module interface and further processing of the non-native polyketide substrate by the ketosynthase (KS) domain. In this study, we aim at disclosing and understanding the low efficiency of chimeric modular PKSs and at establishing guidelines for modular PKSs engineering. To do so, we work with a bimodular PKS testbed and systematically vary substrate specificity, substrate identity, and domain:domain interfaces of the KS involved reactions. We observe that KS domains employed in our chimeric bimodular PKSs are bottlenecks with regards to both substrate specificity as well as interaction with the ACP. Overall, our systematic study can explain in quantitative terms why early oversimplified engineering strategies based on the plain shuffling of modules mostly failed and why more recent approaches show improved success rates. We moreover identify two mutations of the KS domain that significantly increased turnover rates in chimeric systems and interpret this finding in mechanistic detail.

biochemistry↗

bioRxiv2019

The resolution revolution in cryoEM requires new sample preparation procedures: A rapid pipeline to high resolution maps of yeast FAS

Single-particle electron cryo-microscopy (cryoEM) has undergone a \"resolution revolution\" that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 [A] map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.

biochemistry↗

bioRxiv2019

Type I fatty acid synthase (FAS) trapped in the octanoyl-bound state

De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT) and the condensation (KS) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.\n\nHighlightsO_LIThe X-ray structure of the KS-MAT didomain of murine type I FAS is presented in an octanoyl-bound state.\nC_LIO_LIMultiple conformations of the MAT domain and a dynamic active site pocket explain substrate promiscuity.\nC_LIO_LIThe rigid domain structure and minor structural changes upon acylation are in line with the strict substrate specificity of the KS domain.\nC_LIO_LIEnzyme kinetics reveals cooperativity in the KS-mediated transacylation step.\nC_LI

biochemistry↗

bioRxiv2018

Engineering of chimeric polyketide synthases using SYNZIP docking domains

Engineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over twenty years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized, and if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a non-covalent docking domain- or SYNZIP domain-mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a non-covalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced non-covalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.

biochemistry↗

bioRxiv2018

The deadly touch: protein denaturation at the water-air interface and how to prevent it

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation of the protein. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions are those facing the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

biophysics↗

bioRxiv2018

Probing the Modularity of Megasynthases by Rational Engineering of a Fatty Acid Synthase (FAS) Type I

Modularity is an aspect of a decomposable system with a coordinating authority that acts as a glue which holds the loosely held components. These multi-component entities (\"modules\") facilitate rewiring into different designs allowing for change. Such modular character is a fundamental property of many biological entities, especially the family of megasynthases such as polyketide synthases (PKSs). The ability of these PKSs to produce diverse product spectra is strongly coupled to their broad architectural modularity. Decoding the molecular basis of modularity, i.e. identifying the folds and domains that comprise the modules as well as understanding constrains of the assembly of modules, is of utmost importance for harnessing megasynthases for the synthesis of designer compounds. In this study, we exploit the close semblance between PKSs and animal FAS to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by structural and sequence information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel engineered modules resemble all four common module types of PKSs and demonstrate that this approach can be a powerful tool to create higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design.

biochemistry↗

bioRxiv2018

Analysis and Engineering of Substrate Shuttling by the Acyl Carrier Protein (ACP) in Fatty Acid Synthases (FASs)

In the large enzyme complexes of natural biosynthetic pathways, molecules are assembled like in a factory. Carrier domains shuttle substrates and intermediates as covalently attached cargo within the enzyme complex between active sites. The physical confinement of the reaction increases reaction rates and hinders pathway branching. Alternating interactions of substrate-loaded carrier domains with different catalytic domains modulate the chemical environment. In this study, we aim at assessing the impact of domain-domain interactions (DDIs) on the reaction progress of a multienzyme type I fatty acid synthase (FAS) in quantitative terms. We modulate DDIs by single interface mutations, and read out the impact on substrate shuttling by recording fatty acid (FA) chain length product spectra and FAS activities. Our data show that even single interface point mutations can severely affect FA synthesis. With molecular dynamics simulations and modeling, we relate the mutation effects to specific alterations in the molecular interaction networks and domain-domain binding energetics. Some of the presented mutations induce the synthesis of short-chain FAs. These compounds are important commodity products and potent precursors for microbial biofuel production.

bioengineering↗

bioRxiv2018

Molecular mechanisms in fungal fatty acid synthase (FAS) assembly

The fungal fatty acid synthase (fFAS) multienzyme is a barrel-shaped 2.6 MDa complex comprising six times eight catalytic domains. Upon barrel-formation, up to several hundred kDa large polypeptides intertwine to bury about 170,000 [A]2 of protein surface. Functional, regulatory and structural data as well as evolutionary aspects of fFAS have been elucidated during the last decades. Notwithstanding a profound knowledge of this protein family, the biogenesis of the elaborate structure remained elusive. Remarkably, experimental data have recently demonstrated that fFAS self-assembles without the assistance of specific factors. Considering the infinitesimal probability that the barrel-shaped complex forms simply by domains approaching in the correct orientation, we were interested in understanding the sequence of events that have to orchestrate fFAS assembly. Here, we show that fFAS attains its quaternary structure along a pathway of successive domain-domain interactions, which is strongly related to the evolutionary development of this protein family. The knowledge on fFAS assembly may pave the way towards antifungal therapy, and further develops fFAS as biofactory in technological applications.

molecular biology↗

bioRxiv2018

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Amber codon suppression is a powerful tool to site-specifically modify proteins to generate novel biophysical probes. Yet, its application on large and complex multidomain proteins is challenging, leading to difficulties during structural and conformational characterization using spectroscopic methods. The animal fatty acid synthase type I is a 540 kDa homodimer displaying large conformational variability. As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of obesity, diabetes and cancer treatment. Substrates and intermediates remain covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic domains by the acyl carrier protein domain. Thus, conformational variability of animal FAS is an essential aspect for fatty acid biosynthesis. We investigate this multidomain protein as a model system for probing amber codon suppression by genetic encoding of non-canonical amino acids. The systematic approach relies on a microplate-based reporter assay of low complexity, that was used for quick screening of suppression conditions. Furthermore, the applicability of the reporter assay is demonstrated by successful upscaling to both full-length constructs and increased expression scale. The obtained fluorescent probes of murine FAS type I could be subjected readily to a conformational analysis using single-molecule fluorescence resonance energy transfer.

biochemistry↗