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Publikationen - Natur- und Wirkstoffchemie

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Publikation

Schicht, M.; Rausch, F.; Finotto, S.; Mathews, M.; Mattil, A.; Schubert, M.; Koch, B.; Traxdorf, M.; Bohr, C.; Worlitzsch, D.; Brandt, W.; Garreis, F.; Sel, S.; Paulsen, F.; Bräuer, L.; SFTA3, a novel protein of the lung: three-dimensional structure, characterisation and immune activation Eur. Respir. J. 44, 447-456, (2014) DOI: 10.1183/09031936.00179813

The lung constantly interacts with numerous pathogens. Thus, complex local immune defence mechanisms are essential to recognise and dispose of these intruders. This work describes the detection, characterisation and three-dimensional structure of a novel protein of the lung (surfactant-associated protein 3 (SFTA3/SP-H)) with putative immunological features. Bioinformatics, biochemical and immunological methods were combined to elucidate the structure and function of SFTA3. The tissue-specific detection and characterisation was performed by using electron microscopy as well as fluorescence imaging. Three-dimensional structure generation and analysis led to the development of specific antibodies and, as a consequence, to the localisation of a novel protein in human lung under consideration of cystic fibrosis, asthma and sepsis. In vitro experiments revealed that lipopolysaccharide induces expression of SFTA3 in the human lung alveolar type II cell line A549. By contrast, the inflammatory cytokines interleukin (IL)-1β and IL-23 inhibit expression of SFTA3 in A549. Sequence- and structure-based prediction analysis indicated that the novel protein is likely to belong to the family of lung surfactant proteins. The results suggest that SFTA3 is an immunoregulatory protein of the lung with relevant protective functions during inflammation at the mucosal sites.
Publikation

Rausch, F.; Schicht, M.; Bräuer, L.; Paulsen, F.; Brandt, W.; Protein modeling and molecular dynamics simulation of the two novel surfactant proteins SP-G and SP-H J. Mol. Model. 20, 2513, (2014) DOI: 10.1007/s00894-014-2513-0

Surfactant proteins are well known from the human lung where they are responsible for the stability and flexibility of the pulmonary surfactant system. They are able to influence the surface tension of the gas–liquid interface specifically by directly interacting with single lipids. This work describes the generation of reliable protein structure models to support the experimental characterization of two novel putative surfactant proteins called SP-G and SP-H. The obtained protein models were complemented by predicted posttranslational modifications and placed in a lipid model system mimicking the pulmonary surface. Molecular dynamics simulations of these protein-lipid systems showed the stability of the protein models and the formation of interactions between protein surface and lipid head groups on an atomic scale. Thereby, interaction interface and strength seem to be dependent on orientation and posttranslational modification of the protein. The here presented modeling was fundamental for experimental localization studies and the simulations showed that SP-G and SP-H are theoretically able to interact with lipid systems and thus are members of the surfactant protein family.
Publikation

Rausch, F.; Brandt, W.; Schicht, M.; Bräuer, L.; Paulsen, F.; Protein modeling and molecular dynamic studies of two new surfactant proteins J. Cheminform. 5, O2, (2013) DOI: 10.1186/1758-2946-5-S1-O2

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Publikation

Rausch, F.; Schicht, M.; Paulsen, F.; Ngueya, I.; Bräuer, L.; Brandt, W.; “SP-G”, a Putative New Surfactant Protein – Tissue Localization and 3D Structure PLOS ONE 7, e47789, (2012) DOI: 10.1371/journal.pone.0047789

Surfactant proteins (SP) are well known from human lung. These proteins assist the formation of a monolayer of surface-active phospholipids at the liquid-air interface of the alveolar lining, play a major role in lowering the surface tension of interfaces, and have functions in innate and adaptive immune defense. During recent years it became obvious that SPs are also part of other tissues and fluids such as tear fluid, gingiva, saliva, the nasolacrimal system, and kidney. Recently, a putative new surfactant protein (SFTA2 or SP-G) was identified, which has no sequence or structural identity to the already know surfactant proteins. In this work, computational chemistry and molecular-biological methods were combined to localize and characterize SP-G. With the help of a protein structure model, specific antibodies were obtained which allowed the detection of SP-G not only on mRNA but also on protein level. The localization of this protein in different human tissues, sequence based prediction tools for posttranslational modifications and molecular dynamic simulations reveal that SP-G has physicochemical properties similar to the already known surfactant proteins B and C. This includes also the possibility of interactions with lipid systems and with that, a potential surface-regulatory feature of SP-G. In conclusion, the results indicate SP-G as a new surfactant protein which represents an until now unknown surfactant protein class.
Publikation

Wessjohann, L.; Zakharova, S.; Schulze, D.; Kufka, J.; Weber, R.; Bräuer, L.; Brandt, W.; Enzymatic C–C-Coupling Prenylation: Bioinformatics – Modelling – Mechanism – Protein-Redesign – Biocatalytic Application Chimia 63, 340-344, (2009) DOI: 10.2533/chimia.2009.340

The functional role of isoprenoids and especially enzymatic prenylation in nature and human application is briefly covered, with the focus on bioinformatical, mechanistical and structural aspects of prenyltransferases and terpene synthases. These enzymes are as yet underrepresented but perspectively useful biocatalysts for C–C couplings of aromatic and isoprenoid substrates. Some examples of the successful use in chemoenzymatic synthesis are given including an application for the otherwise difficult synthesis of Kuhistanol A. Computational structure-based site-directed mutagenesis can be used for rational enzyme redesign to obtain altered substrate and product specificities, which is demonstrated for terpene cyclases.
Publikation

Brandt, W.; Bräuer, L.; Günnewich, N.; Kufka, J.; Rausch, F.; Schulze, D.; Schulze, E.; Weber, R.; Zakharova, S.; Wessjohann, L.; Molecular and structural basis of metabolic diversity mediated by prenyldiphosphate converting enzymes Phytochemistry 70, 1758-1775, (2009) DOI: 10.1016/j.phytochem.2009.09.001

General thermodynamic calculations using the semiempiric PM3 method have led to the conclusion that prenyldiphosphate converting enzymes require at least one divalent metal cation for the activation and cleavage of the diphosphate–prenyl ester bond, or they must provide structural elements for the efficient stabilization of the intermediate prenyl cation. The most important common structural features, which guide the product specificity in both terpene synthases and aromatic prenyl transferases are aromatic amino acid side chains, which stabilize prenyl cations by cation–π interactions. In the case of aromatic prenyl transferases, a proton abstraction from the phenolic hydroxyl group of the second substrate will enhance the electron density in the phenolic ortho-position at which initial prenylation of the aromatic compound usually occurs.A model of the structure of the integral transmembrane-bound aromatic prenyl transferase UbiA was developed, which currently represents the first structural insight into this group of prenylating enzymes with a fold different from most other aromatic prenyl transferases. Based on this model, the structure–activity relationships and mechanistic aspects of related proteins, for example those of Lithospermum erythrorhizon or the enzyme AuaA from Stigmatella aurantiaca involved in the aurachin biosynthesis, were elucidated. The high similarity of this group of aromatic prenyltransferases to 5-epi-aristolochene synthase is an indication of an evolutionary relationship with terpene synthases (cyclases). This is further supported by the conserved DxxxD motif found in both protein families. In contrast, there is no such relationship to the aromatic prenyl transferases with an ABBA-fold, such as NphB, or to any other known family of prenyl converting enzymes. Therefore, it is possible that these two groups might have different evolutionary ancestors.
Publikation

Bräuer, L.; Brandt, W.; Schulze, D.; Zakharova, S.; Wessjohann, L.; A Structural Model of the Membrane-Bound Aromatic Prenyltransferase UbiA from E. coli ChemBioChem 9, 982-992, (2008) DOI: 10.1002/cbic.200700575

We report the first reasonable model for the active site of the membrane‐bound aromatic prenyltransferase UbiA, derived from structural—not sequence—similarity to a terpene synthase, with the aid of threading, site‐directed mutagenesis, and substrate selectivities. The high similarity of the active fold of UbiA‐transferase to that of 5‐epi‐aristolochene synthase (Nictotiana tabacum ), despite a low homology, allows a hypothesis on a convergent evolution of these enzymes to be formed.
Publikation

Bräuer, L.; Brandt, W.; Wessjohann, L. A.; Modeling the E. coli 4-hydroxybenzoic acid oligoprenyltransferase (ubiA transferase) and characterization of potential active sites J. Mol. Model. 10, 317-327, (2004) DOI: 10.1007/s00894-004-0197-6

4-Hydroxybenzoate oligoprenyltransferase of E. coli, encoded in the gene ubiA, is an important key enzyme in the biosynthetic pathway to ubiquinone. It catalyzes the prenylation of 4-hydroxybenzoic acid in position 3 using an oligoprenyl diphosphate as a second substrate. Up to now, no X-ray structure of this oligoprenyltransferase or any structurally related enzyme is known. Knowledge of the tertiary structure and possible active sites is, however, essential for understanding the catalysis mechanism and the substrate specificity.With homology modeling techniques, secondary structure prediction tools, molecular dynamics simulations, and energy optimizations, a model with two putative active sites could be created and refined. One active site selected to be the most likely one for the docking of oligoprenyl diphosphate and 4-hydroxybenzoic acid is located near the N-terminus of the enzyme. It is widely accepted that residues forming an active site are usually evolutionary conserved within a family of enzymes. Multiple alignments of a multitude of related proteins clearly showed 100% conservation of the amino acid residues that form the first putative active site and therefore strongly support this hypothesis. However, an additional highly conserved region in the amino acid sequence of the ubiA enzyme could be detected, which also can be considered a putative (or rudimentary) active site. This site is characterized by a high sequence similarity to the aforementioned site and may give some hints regarding the evolutionary origin of the ubiA enzyme.Semiempirical quantum mechanical PM3 calculations have been performed to investigate the thermodynamics and kinetics of the catalysis mechanism. These results suggest a near SN1 mechanism for the cleavage of the diphosphate ion from the isoprenyl unit. The 4-hydroxybenzoic acid interestingly appears not to be activated as benzoate anion but rather as phenolate anion to allow attack of the isoprenyl cation to the phenolate, which appeared to be the rate limiting step of the whole process according to our quantum chemical calculations. Our models are a basis for developing inhibitors of this enzyme, which is crucial for bacterial aerobic metabolism.
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