The Function of the Halophilic Dodecin

Flavins are physiologically relevant cofactors that catalyze various redox and light-induced reactions. Due to a high intrinsic reactivity, these compounds are found tightly bound to proteins with the chemistry of the flavin either narrowed to a defined reaction channel (flavoenzymes) or reduced to (almost) non-reactivity (flavin binding and carrier proteins). Lumichrome is a product of flavin photodegradation. In spite of the structural similarity to flavins, lumichrome has electronic properties which differ from flavins, preventing this compound from any physiological relevance as a cofactor. The interest in lumichrome is basically focussed on its role as a photosensitizing compound. Lumichrome is excited by the absorption of visible light and relaxes by transferring electrons or electronic energy to surrounding substrates and oxygen, exerting an unspecific toxic effect on the cellular environment. Dodecin is a dodecameric flavin binding protein comprising a novel ligand binding fold. It incorporates dimers of ligands arranged in antiparallel manner within each of the six identical binding pockets. In this thesis, structure and function of dodecin from the archaeal organism Halobacterium salinarum are reported. X-ray structural investigations supplemented with functional data revealed that this protein is an unspecific binder of flavins and binder of the flavin-like compound lumichrome. Dissociation constants were obtained in the nanomolar to micromolar range and found to correlate positively with the ligand size. The preference of dodecin for the small ligands lumichrome and lumiflavin is described as a gated ligand binding mode, based on the low plasticity of the dodecin binding pocket which sterically restricts the bulkier ligands from arranging the flavin aromatic subunit in a high affinity position. Site directed mutagenesis of the halophilic dodecin allowed to spread the idea of dodecin as a small ligand binding particle among homologous proteins. These mutational studies could moreover show that the halophilic type of dodecins is outstanding in additionally exhibiting a high affinity for riboflavin. The stabilization of the ribityl chain by an H-bond network to a single residue was found to suspend restrictions of the gated ligand binding mode and to enable H. salinarum dodecin to exhibit multiple (high) affinity. In Western-Blot and RT-PCR analysis of the dodecin expression level, it could be demonstrated that after a short lag period dodecin is constitutively expressed in light and in dark. In the late stationary phase, a clear influence of dodecin on the riboflavin cellular concentrations could be observed. While high levels of riboflavin were found in H. salinarum wild type cells, in cells of the dodecin deficient strain riboflavin cellular concentrations were depressed. Lumichrome concentrations on the other hand were unaffected from dodecin; however; increased concentrations of lumichrome were found in light, according to a photolytic degradation of riboflavin. In vivo data fully agreed with the deductions from the dodecin structural and functional investigations. Dodecin is a riboflavin binding and carrier protein (RfBP). Its function is to store riboflavin under non-favorable environmental conditions while preventing this flavin from photodegradation. The lumichrome-collecting property represents an extra-feature which allows binding of lumichrome if degradation of riboflavin occurs in order to protect the cellular environment from high amounts of this photo-toxic compound

The Function of the Halophilic Dodecin

Flavins are physiologically relevant cofactors that catalyze various redox and light-induced reactions. Due to a high intrinsic reactivity, these compounds are found tightly bound to proteins with the chemistry of the flavin either narrowed to a defined reaction channel (flavoenzymes) or reduced to (almost) non-reactivity (flavin binding and carrier proteins). Lumichrome is a product of flavin photodegradation. In spite of the structural similarity to flavins, lumichrome has electronic properties which differ from flavins, preventing this compound from any physiological relevance as a cofactor. The interest in lumichrome is basically focussed on its role as a photosensitizing compound. Lumichrome is excited by the absorption of visible light and relaxes by transferring electrons or electronic energy to surrounding substrates and oxygen, exerting an unspecific toxic effect on the cellular environment. Dodecin is a dodecameric flavin binding protein comprising a novel ligand binding fold. It incorporates dimers of ligands arranged in antiparallel manner within each of the six identical binding pockets. In this thesis, structure and function of dodecin from the archaeal organism Halobacterium salinarum are reported. X-ray structural investigations supplemented with functional data revealed that this protein is an unspecific binder of flavins and binder of the flavin-like compound lumichrome. Dissociation constants were obtained in the nanomolar to micromolar range and found to correlate positively with the ligand size. The preference of dodecin for the small ligands lumichrome and lumiflavin is described as a gated ligand binding mode, based on the low plasticity of the dodecin binding pocket which sterically restricts the bulkier ligands from arranging the flavin aromatic subunit in a high affinity position. Site directed mutagenesis of the halophilic dodecin allowed to spread the idea of dodecin as a small ligand binding particle among homologous proteins. These mutational studies could moreover show that the halophilic type of dodecins is outstanding in additionally exhibiting a high affinity for riboflavin. The stabilization of the ribityl chain by an H-bond network to a single residue was found to suspend restrictions of the gated ligand binding mode and to enable H. salinarum dodecin to exhibit multiple (high) affinity. In Western-Blot and RT-PCR analysis of the dodecin expression level, it could be demonstrated that after a short lag period dodecin is constitutively expressed in light and in dark. In the late stationary phase, a clear influence of dodecin on the riboflavin cellular concentrations could be observed. While high levels of riboflavin were found in H. salinarum wild type cells, in cells of the dodecin deficient strain riboflavin cellular concentrations were depressed. Lumichrome concentrations on the other hand were unaffected from dodecin; however; increased concentrations of lumichrome were found in light, according to a photolytic degradation of riboflavin. In vivo data fully agreed with the deductions from the dodecin structural and functional investigations. Dodecin is a riboflavin binding and carrier protein (RfBP). Its function is to store riboflavin under non-favorable environmental conditions while preventing this flavin from photodegradation. The lumichrome-collecting property represents an extra-feature which allows binding of lumichrome if degradation of riboflavin occurs in order to protect the cellular environment from high amounts of this photo-toxic compound

Betriebliche Gesundheitsförderung in oberösterreichischen Klein- und Mittelbetrieben

Die vorliegende Arbeit befasst sich mit Betrieblicher Gesundheitsförderung in Klein- und Mittelbetrieben. Es soll veranschaulicht werden welche Entwicklungen in der Arbeitswelt dieses Thema in Zukunft immer wichtiger werden lässt. Mit einer Befragung in diversen oberösterreichischen Unternehmen, soll zusätzlich ermittelt werden ob das Thema der Gesundheitsförderung bei den Mitarbeitern bekannt ist und ob Maßnahmen zur Gesundheitsförderung angeboten werden, bzw. in welchen Bereichen Bedarf besteht. Anhand eines Beispieles wird ein Konzept für ein Gesundheitsförderungsprogramm, das speziell auf die Bedürfnisse kleiner Unternehmen zugeschnitten ist, vorgestellt

Structure of the archaeal chemotaxis protein CheY in a domain-swapped dimeric conformation

Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection

Structural insights into the mechanism of archaellar rotational switching

Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum. Signal transduction via phosphorylated CheY is conserved in bacteria and archaea. In this study, the authors employ structural biochemistry combined with cell biology to delineate the mechanism of CheY recognition by the adaptor protein CheF

Strategies in megasynthase engineering – fatty acid synthases (FAS) as model proteins

Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the "assembly-line synthetic concept" of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field

Transacylation kinetics in fatty acid and polyketide synthases and its sensitivity to point mutations

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

Correction: Engineering strategies for rational polyketide synthase design

The authors regret that there is an error present in the units displayed in the sentence “The dissociation constant of docking domains or modules connected by docking domains was found to be KD 70–130 mM (ref. 35) and KD 1–2 mM (ref. 59), respectively.” within Section 3.1. Module–module exchanges. The corrected version of this sentence is as follows: The dissociation constant of docking domains or modules connected by docking domains was found to be KD 70–130 μM (ref. 35) and KD 1–2 mM (ref. 59), respectively. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers

Engineering strategies for rational polyketide synthase design

Covering: mid 1990s to 2018 Over the last two decades, diverse approaches have been explored to generate new polyketides by engineering polyketide synthases (PKSs). Although it has been proven possible to produce new compounds by designed PKSs, engineering strategies failed to make polyketides available via widely applicable rules and protocols. Still, organic synthetic routes have to be employed whenever new polyketides are needed for applications in medicine, agriculture, and industry. In light of the rising demand for commodity products from feedstock and for fast and cheap access to pharmaceutical compounds, the need for harnessing PKSs to produce such molecules is more urgent than ever before. In this review, we focus on a multitude of approaches to engineer modular PKSs by swapping and replacing PKS modules and domains, which we analyze in the light of recent structural and biochemical data. We conclude with an outlook on possible strategies on how to increase success rates of PKS engineering in future