• Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa.

      Hagelueken, Gregor; Wiehlmann, Lutz; Adams, Thorsten M; Kolmar, Harald; Heinz, Dirk W; Tümmler, Burkhard; Schubert, Wolf-Dieter; Molecular Host-Pathogen Interactions, Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2007-07-24)
      Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon metabolism to the membrane-bound alkane hydroxylases AlkB1 and AlkB2. The RdxR-Rdx system also is crucial as part of the oxidative stress response in archaea or anaerobic bacteria. The redox couple has been analyzed in detail as a model system for ET processes. We have solved the structure of RdxR of P. aeruginosa both alone and in complex with Rdx, without the need for cross-linking, and both structures were refined at 2.40- and 2.45-A resolution, respectively. RdxR consists of two cofactor-binding domains and a C-terminal domain essential for the specific recognition of Rdx. Only a small number of direct interactions govern mutual recognition of RdxR and Rdx, corroborating the transient nature of the complex. The shortest distance between the redox centers is observed to be 6.2 A.
    • Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III).

      Glotzbach, Bernhard; Schmelz, Stefan; Reinwarth, Michael; Christmann, Andreas; Heinz, Dirk W; Kolmar, Harald; Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Germany. (2013-01)
      In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.