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The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel `hairpin-like' architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the `hairpin-like' hypothesis. [ABSTRACT FROM AUTHOR]

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2 S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2 S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific 1H/2H isotope exchange observed by 1H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1. [ABSTRACT FROM AUTHOR]

Chiral amines are important precursors for the pharmaceutical and fine-chemical industries. Because of this, the demand for enantiopure amines is currently increasing. Amine transaminases can produce a large spectrum of chiral amines in the ( R)- or ( S)-configuration, depending on their substrate scope and stereo-preference, by converting a prochiral ketone into the chiral amine while using alanine as the amine donor producing pyruvate as an α-keto acid product. In order to guide the protein engineering of transaminases to improve substrate specificity and enantioselectivity, we carried out a crystal structure analysis at 1.6 Å resolution of the ( R)-amine transaminase from Aspergillus fumigatus with the bound inhibitor gabaculine. This revealed that Arg126 has an important role in the dual substrate recognition of this enzyme because mutating this residue to alanine reduced substantially the ability of the enzyme to use pyruvate as an amino acceptor. Database Coordinates and structure factors have been deposited with the Protein Data Bank under accession code . [ABSTRACT FROM AUTHOR]

The importance of amine transaminases for producing optically pure chiral precursors for pharmaceuticals and chemicals has substantially increased in recent years. The X-ray crystal structure of the (R) -selective amine transaminase from the fungus Aspergillus fumigatus was solved by S-SAD phasing to 1.84 Å resolution. The refined structure at 1.27 A resolution provides detailed knowledge about the molecular basis of substrate recognition and conversion to facilitate protein-engineering approaches. The protein forms a homodimer and belongs to fold class IV of the pyridoxal-5'-phosphate-dependent enzymes. Both subunits contribute residues to form two active sites. The structure of the holoenzyme shows the catalytically important cofactor pyridoxal-5'-phosphate bound as an internal aldimine with the catalytically responsible amino-acid residue Lys179, as well as in its free form. A long N-terminal helix is an important feature for the stability of this fungal (R)-selective amine transaminase, but is missing in branched-chain amino-acid aminotransferases and d-amino-acid aminotransferases. [ABSTRACT FROM AUTHOR]

Flavonoide stellen eine große Gruppe pflanzlicher Sekundärmetabolite dar und haben eine Vielzahl unterschiedlicher biologischer Eigenschaften. Aufgrund dessen stehen sie im Interesse vieler Wissenschaftler, da sie zu vielen industriell interessanten Verbindungen führen können. Das anaerobe Darmbakterium Eubacterium ramulus kann über einen bislang unbekannten Abbauweg diese Flavonoide verstoffwechseln. Über eine Vollgenom ‐ Sequenzierung wurden in dieser Arbeit die entscheidenden Enzyme, eine Chalconisomerase (CHI) und eine Enoatreduktase (ERED), identifiziert, kloniert und funktionell sogar unter aeroben Bedingungen exprimiert. Mittels Biotransformation des Flavanons zum Dihydrochalcon konnte der Flavonoid ‐ Abbauweg bewiesen werden. Der beide Enzyme exprimierende E. ‐ coli ‐ Stamm kann für die Umsetzung verschiedener Flavanone eingesetzt werden. Dies unterstreicht die Anwendbarkeit des in dieser Arbeit entwickelten biokatalytischen Systems. Der biokatalytisch ‐ metabolische Weg für die Umsetzung von Flavonoiden aus Eubacterium ramulus wurde identifiziert. Eine Chalconisomerase und eine Enoatreduktase wurden erfolgreich kloniert und in E. coli exprimiert und unter aeroben Bedingungen eingesetzt, obwohl E. ramulus ein strikt anaerobes Bakterium ist. Der beide Enzyme exprimierende E. ‐ coli ‐ Stamm kann für die Umsetzung verschiedener Flavanone eingesetzt werden. [ABSTRACT FROM AUTHOR]

Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction. [ABSTRACT FROM AUTHOR]

In an extracorporeal combination therapy, the impact of different replacement fluids on survival was tested in a bacterial sepsis model in pigs. In an animal study 19 pigs, weighing 7.5-11.1 kg, were included. All groups received an intravenous lethal dose of live Staphylococcus aureus over 1 h. The animals were treated by an extracorporeal circuit consisting of online centrifugation and subsequent plasma filtration for 4 h. The extracorporeal circuit was pre-filled with 400 mL replacement fluid. In the P0 group 100% hydroxyethyl starch 130/0.4 was used as replacement fluid; in the P30 group 30% pig plasma and 70% hydroxyethyl starch; and in the P100 group 100% pig plasma. The observation time was 7 days. All animals of the group P100 survived, while all animals of group P0 and five out of seven animals of the P30 group died during the observation time. Extracorporeal therapy consisting of online centrifugation and plasma filtration with 100% pig plasma as replacement fluid significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged. [ABSTRACT FROM AUTHOR]

The impact on survival of a combination of plasma separation by centrifugation and subsequent plasma filtration was tested in a bacterial sepsis model in pigs. In this animal study 19 pigs were included. Groups II and III received an intravenous lethal dose of live Staphylococcus aureus over 1 h; group I received saline (non-septic control-NC). Groups I and II were treated by an extracorporeal circuit consisting of online centrifugation and subsequent plasma filtration (group II: treated group-TG) for 4 h; group III had no specific treatment (septic control, SC). The observation time was 7 days. All animals of group I (NC) and group II (TG) survived, while all animals of group III (SC) died during the observation time. Extracorporeal therapy with online centrifugation and plasma filtration significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged. [ABSTRACT FROM AUTHOR]

The ( R)-selective amine transaminase from Aspergillus fumigatus was expressed in Escherichia coli and purified to homogeneity. Bright yellow crystals appeared while storing the concentrated solution in the refrigerator and belonged to space group C2221. X-ray diffraction data were collected to 1.27 Å resolution, as well as an anomalous data set to 1.84 Å resolution that was suitable for S-SAD phasing. [ABSTRACT FROM AUTHOR]