Biochemistry

At the University facilities

Research Interests

Bioenergetics with emphasis on the assembly, structure and function of the proton-pumping NADH:ubiquinone oxidoreductase of mitochondria and purple bacteria using Neurospora crassa and Escherichia coli as model organisms.

The fatty acid synthetic system of mitochondria; identification of the products of this system; isolation and characterisation of enzymes involved in this system.

Metabolic design in filamentous fungi; the role of mitochondrial respiration in the overproduction of primary metabolites in Aspergillus niger.

Neurodegenerative diseases associated with defects in the mitochondrial respiratory chain: Morbus Parkinson, idiopathic dystonia.

Funding

DFG Sonderforschungsbereich 189 "Differenzierung und Regulation energieumwandelnder biologischer Systeme", the DFG Forschergruppe "Molekularbiologie neurogegenarativer Erkrankungen", and the BMFT Schwerpunktprojekt "Stoffumwandlung mit Biokatalysatoren".

Resources and Equipment

Good equippment and experience exist for molecular genetic studies with Escherichia coli, Neurospora crassa and Aspergillus niger, for membrane protein isolation and analysis including steady-state and pre-steady-state analysis of electron transfer enzmyes. The institute has a new x-band EPR-spectrometer equipped with a liquid helium cooling system.

There are some 10 diploma and some 15 doctoral students and 4 postdoctoral research workers in the institute of biochemistry.

At the facilities of the Forschungszentrum Jülich GmbH

Research Interests

Structure and function of carrier systems for amino acids and other metabolites in bacteria, molecular analysis, physiology and regulation of their function.

Relevance of amino acid excretion in Gram-positive (Corynebacterium glutamicum) and Gram-negative (E. coli) bacteria for the biotechnological production of amino acids.

Analysis of structure and function of mitochondrial carrier proteins in reconstituted systems.

Funding

Deutsche Forschungsgemeinschaft (Research Council),Bundesministerium für Bildung, Wissenschaft und Forschung,European Community, Joint projects with several companies

Resources and Equipment

Modern biochemical equipment (HPLC, FPLC, etc.); DNA sequence analyzer and oligonucleotide synthesizer; Small, medium and large scale fermentation facilities

Staff Research Interest

Prof. Dr. Hanns Weiss (Head of section)

Investigation of the enzymes and the products of the mitochondrial fatty acid synthetic
system;

Structure and function of the mitochondrial proton-pumping NADH:ubiquinone
oxidoreductase (respiratory complex I). For both subjects Neurospora crassa is used as
model organism.

Prof. Dr. Reinhard Krämer (Facility of Forschungszentrum Jülich, GmbH)

Structure and function of bacterial transport systems

Biotechnology and biochemistry of amino acid production by bacteria

Structure and function of mitochondrial carrier proteins

Dr. Ulrich Schulte

Biogenesis of the proton-pumping NADH:ubiquinone oxidoreductase (complex I)
from Neurospora crassa

Dr. Thorsten Friedrich

Structure and mechanism of the proton-pumping NADH:ubiquinone oxidoreductase
(complex I) from Escherichia coli

EPR spectroscopy

HPLC

Enzyme kinetics

Dr. Götz Hofhaus

Defects in the mitochondrial respiratory chain associated with neurodegenerative diseases
in humans like Morbus Parkinson and idopathic dystonia.

Dr. Regina Schneider

Mitochondrial fatty acid synthesis

The role of mitochondrial respiration in the overproduction of primary metabolites in Aspergillus niger

Prof. Dr. Hanns Weiss

Status: Chair of the Institute of Biochemistry

Tel: +49 211 311 4950

Fax: +49 211 311 3085

Postal address: Heinrich-Heine-Universität Düsseldorf

Institut für Biochemie

Universitätsstraße 1

40225 Düsseldorf

email:. Hanns.Weiss.@uni-duesseldorf.de

Research interests:

The role of the mitochondrial fatty acid synthesis.

The proton-pumping respiratory complex I of bacteria and mitochondria and its homologue of chloroplasts

THE ROLE OF THE MITOCHONDRIAL FATTY ACID SYNTHESIS

De novo fatty acid synthesis is performed in all species by the same sequence of enzymic reactions. The structure and location of the enzymes catalysing the reactions, however, differ among different species. In bacteria, the fatty acid synthase (FAS) of type II consists of seven discrete polypeptides. Plants contain a similar FAS type II in their chloroplasts. In fungi, all enzyme activities are domains of two polypeptides, and in animals of a single polypeptide. This eucaryotic FAS type I is localized in the cytosol.

Discovery of proteins in mitochondria homologous to enzymes involved in bacterial fatty acid synthesis gives rise to the suggestion that a so far unknown synthetic pathway leading to fatty acids is localized also in this organell of eucaryotic cells. The investigation of phenotypes of mutants in which the genes encoding these proteins were disrupted shows that this synthetic pathway is important for oxidative phosphorylation.

References

Sackmann, U., Zensen, R., Röhlen, D., Jahnke, U. and Weiss, H. (1991) The mitochondrial acyl-carrier protein is a subunit of NADH:ubiquinone reductase (complex I) in Neurospora crassa Eur. J. Biochem. 200, 463-469.

Zensen, R., Husmann, H., Schneider, R., Friedrich, T., Peine, T., and Weiss, H (1992). De novo synthesis and desaturation of fatty acids at the mitochondrial acyl-carrier protein, a subunit of NADH:ubiquinone oxidoreductase in Neurospora crassa FEBS Lett. 310, 179-182.

Schneider, R., Massow, M. Lisowsky, T. and Weiss, H. (1995) Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisae after inactivation of the gene encoding the mitochondrial acyl carrier protein. Curr. Genetics, in press.

THE PROTON-PUMPING RESPIRATORY COMPLEX I OF BACTERIA AND MITOCHONDRIA AND ITS HOMOLOGUE OF CHLOROPLASTS

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first of the respiratory complexes providing the proton motive force which is essential for the synthesis of ATP. Closely related forms of this complex exist in the mitochondria of eucaryotes and in the plasma membranes of purple bacteria. The minimal structural framework common to the mitochondrial and the bacterial complex is composed of 14 polypeptides with 1 FMN and 6-8 iron-sulfur clusters as prosthetic groups. The mitochondrial complex contains many accessory subunits for which no homologous counterparts exist in the bacterial complex. Genes for 11 of the 14 minimal subunits are also found in the plastidal DNA of plants and in the genome of cyanobacteria. However, genes encoding subunits of the NADH dehydrogenase part of complex I are apparently missing in these species. The possibility is discussed that chloroplasts and cyanobacteria contain a complex I equiped with a different electron input device. This complex may work as a NAD(P)H: or a ferredoxin:plastoquinone oxidoreductase participating in cyclic electron transport during photosynthesis.

References

Weiss, H., Friedrich, T., Hofhaus, G. and Preis, D. (1991): The respiratory chain NADH dehydrogenase (Complex I) of mitochondria. Eur. J. Biochem. (Review) 197, 563-576.

Weiss, H. and Friedrich T. (1991): Redox-linked proton translocation by NADH-ubiquinone reductase J. Bioenerget. Biomembrane 23, 743-754.

Hofhaus, G., Weiss, H. and Leonard, K. (1991): Electron microscopic analysis of the peripheral and the membrane parts of mitochondrial NADH dehydrogenase (Complex I). J. Mol. Biol. 221, 1027-1043.

Nehls, U., Friedrich, T., Schmiede , A., Ohnishi, T. and Weiss, H (1992). Characterisation of assembly intermediates of NADH:ubiquinone oxidoreductase (complex I) accumulated in Neurospora mitochondria by gene disruption J. Mol. Biol. 227, 1032-1042.

Weidner, U., Geier, S., Ptock, A., Friedrich, T., Leif, H. and Weiss, H (1993): The gene locus of the proton translocating NADH:ubiquinone oxidoreductase in Escherichia coli: organisation of the 14 genes and relationship of the derived proteins with subunits of mitochondrial complex I J. Mol. Biol. 233, 109-122.

Leif, H., Sled, V.D., Ohnishi, T., Weiss, H. and Friedrich, T. (1995) Isolation and characterization of the proton-translocating NADH:ubiquinone oxidoreductase from Escherichia coli. Eur. J. Biochem, 230, 538-548.

Schulte, U. & Weiss, H. (1995) NADH-ubiquinone reductase (Complex I) from N. crassa. In "Mitochondrial genetics and Biogenesis" (G. Attardi and A. Chomyn eds.) Meth. Enzymol., submitted.

Friedrich, T., Steinmüller, K. & Weiss, H. (1995) The proton-pumping respiratory complex I of bacteria and mitochondria and is homologue of chloroplasts. FEBS Lett. (Minireview), 367, 107-111.

Prof. Dr. Reinhard Krämer

Status: Professor at the Institute of Biotechnology I

Tel: +49 2461 61 55 15

Postal address: Forschungszentrum Jülich

Institut für Biotechnologie I

Postfach 1913

52425 Jülich

Research interests:

Bacterial Amino Acid Carriers

Bacterial Carriers for Other Solutes

Mitochondrial Carrier Proteins

Bacterial Amino Acid Carriers

Bacteria are used worldwide for amino acid production (e.g. glutamate, lysine, threonine, aromatic amino acids). We have characterized a number of specific, energy dependent, tightly regulated carrier systems responsible for the excretion of these amino acids by bacteria under conditions of amino acid production [2-4]. These systems have been shown to be present both in Corynebacterium glutamicum and in Escherichia coli, their activity may be limiting in production processes [5]. They are regulated on the level of expression and activity and are integrated into the networks of metabolic regulation. We have also elucidated the uptake systems for these amino acids, since their activity has to be coordinated with that of the corresponding excretion systems [2-4].

References

[1] Krämer, R. (1994) Biochim. Biophys. Acta 1185:1-34

[2] Krämer, R. (1994) FEMS Microbiol. Rev. 13:75-93

[3] Sahm, H., Eggeling, L., Eikmanns, B. and Krämer, R. (1995) FEMS Microbiol. Rev. 16:243-

252

[4] Gutmann, M., Hoischen, C. and Krämer, R. (1992) Biochim. Biophys. Acta 1112:115-123

[5] Bröer, S., Eggeling, L. and Krämer, R. (1993) Appl. Environm. Microbiol. 59:316-321

Bacterial Carriers for Other Solutes

We have selected a number of other transport processes in various bacteria (C. glutamicum, E. coli, Zymomonas mobilis) and fungi in order to study important functional and metabolic aspects of carrier systems. Transport systems related to osmoregulation, e.g. glycine betaine uptake [6], to nitrogen metabolism, e.g. ammonia or urea uptake, as well as sugar uptake [7] are investigated.

References

[1] Krämer, R. (1994) Biochim. Biophys. Acta 1185:1-34

[6] Farwick, M., Siewe, R. and Krämer, R. (1995) J. Bacteriol, in press

[7] Weisser, P., Krämer, R., Sahm, H. and Sprenger, G.A. (1995) J. Bacteriol. 177:3351-3354

Mitochondrial Carrier Proteins

The inner mitochondrial membrane carries a set of different transport proteins important for energy metabolism as well as other catabolic and anabolic functions [8]. We use isolated and purified carrier proteins from the inner mitochondrial membrane (e.g. aspartate/glutamate- and phosphate carrier) to study their function after incorporation into artificial phospholipid membranes (liposomes and lipid bilayers) [9]. The structure-function correlation of transport proteins is investigated by using site-specific mutants of the mitochondrial phosphate carrier from bakers yeast (Saccharomyces cerevisiae), heterologously expressed in E. coli.

References

[1] Krämer, R. (1994) Biochim. Biophys. Acta 1185:1-34

[8] Palmieri, R., Indiveri, C. Bisaccia, F. and Krämer, R. (1993) J. Bioenerg. Biomembrane

25:525-535

[9] Stappen, R. and Krämer, R. (1994) J. Biol. Chem. 269:11240-11246

Dr. Ulrich Schulte

Status: Akadem. Rat

Tel: +49 (0)211 311 2020

Fax: +49 (0)211 311 3085

Postal address: Heinrich-Heine-Universitaet Duesseldorf,

Institut für Biochemie

Universitaetsstr.1

40225 Duesseldorf

email: ulrich.schulte@uni-duesseldorf.de

Research interests:

Biogenesis of the proton-pumping NADH:ubiquinone oxidoreductase (complex I)
from Neurospora crassa

BIOGENESIS OF THE PROTON-PUMPING NADH:UBIQUINONE OXIDOREDUCTASE (COMPLEX I) FROM NEUROSPORA CRASSA

The subunits of the mitochondrial complex I are encoded by two different genetic systems. While the majority are nuclear-encoded, synthesized in the cytoplasm and imported into the mitochondrion posttranslationally, the 7 most hydrophobic subunits are encoded and synthesized within the mitochondrion. Assembly of the complex involves formation of fairly stable intermediates, which join like building blocks to yield mature enzyme. We aim to analyse the assembly process and to identify additional proteins involved. The experimental approach includes construction and characterisation of mutants, pulse-chase labeling, and isolation and characterisation of assembly intermediates.

References

Tuschen, G., Sackmann, U., Nehls, U., Haiker, H., Buse, G., and Weiss, H. (1990) Assembly of NADH:ubiquinone reductase (complex I) in Neurospora mitochondria: Independent pathways of nuclear encoded and mitochondrially encoded subunits. J. Mol. Biol. 213, 845-857.

Nehls, U., Friedrich, T., Schmiede, A., Ohnishi, T., and Weiss, H. (1992) Characterisation of assembly intermediates of NADH:ubiquinone oxidoreductase (complex I) accumulated in Neurospora by gene disruption. J. Mol. Biol. 227, 1032-1042.

Schmidt, M., Friedrich, T., Wallrath, J., Ohnishi, T., and Weiss, H. (1992) Accumulation of the preassembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese-limited grown Neurospora crassa. FEBS Lett. 313, 8-11.

Schulte, U., Fecke, W., Krüll, C., Nehls, U., Schmiede, A., Schneider, R., Ohnishi, T., and Weiss, H. (1994) In vivo dissection of the mitochondrial respiratory NADH:ubiquinone oxidoreductase (complex I). Biochim. Biophys. Acta 1187, 121-124.

Schulte, U. and Weiss, H. (1995) Generation and characterization of NADH:ubiquinone oxidoreductase mutants in Neurospora crassa. Meth. Enzymol. 260, 3-14.

Dr. Thorsten Friedrich

Status: Research Assistent, Group Leader

Tel: +49 (0)211 311 2647

Fax: +49 (0)211 311 3085

Postal address: Heinrich-Heine-Universitaet Duesseldorf

Institut für Biochemie

Universitaetsstr.1

40225 Duesseldorf

email: Thorsten.Friedrich@uni-duesseldorf.de

Research interests:

Structure and mechanism of the proton-pumping NADH:ubiquinone oxidoreductase (complex I)
from Escherichia coli

EPR spectroscopy

HPLC

Enzyme kinetics

STRUCTURE AND MECHANISM OF THE PROTON-PUMPING NADH:UBIQUINONE OXIDOREDUCTASE (COMPLEX I) FROM ESCHERICHIA COLI

The respiratory NADH:ubiquinone oxidoreductase, also called complex I, links the electron transfer from NADH to ubiquinone with the translocation of protons across the mitochondrial inner membrane. One FMN and 6-8 iron-sulfur (FeS) clusters serve as prosthetic groups in this reaction. The mitochondrial complex contains some 40 different subunits. Most of them are nuclear-encoded and made in the cytoplasm, whereas the 7 most hydrophobic subunits are encoded and synthesized within the mitochondrion. The nuo operon (from NADH:ubiquinone oxidoreductase) of E. coli encodes 14 proteins designated NuoA - N of which 7 are homologous to nuclear-encoded and 7 to mitochondrially encoded subunits of the eucaryotic complex I. These 14 subunits include all subunits presumed to bind the substrates and to harbour the redox groups. It appears that the nuo operon of E. coli encodes a minimal form of complex I. The isolated entire E. coli complex I contains the 14 proteins encoded by the nuo operon. The preparation contains 1 FMN and at least 3 binuclear (N1a, N1b and N1c) and 3 tetranuclear (N2, N3 and N4) iron-sulfur clusters. It can easily be dissected into three distinct fragments still bearing their redox groups. The subunit composition of these fragments correlates to the clustering of the genes in the nuo operon. Based on the fragmentation pattern and on the EPR spectroscopic analysis, a topological model for the complex was developed.

References

Weiss et al., Eur.J.Biochem. 197, 563-576 (1991)

Weiss and Friedrich, J.Bioenerg.Biomembr. 23, 743-754, (1991)

Weidner et al., J.Mol.Biol. 233, 109-122, (1993)

Sled' et al., J.Bioenerg.Biomembr. 25, 347-356, (1993)

Friedrich et al., J.Bioenerg.Biomembr. 233, 331-337, (1993)

Friedrich et al., Eur.J.Biochem. 219, 691-698, (1994)

Leif et al., Eur.J.Biochem. 230, 538-548 (1995)

Friedrich et al., FEBS Lett. 367, 107-11 (1995)

Dr. Götz Hofhaus

Status: Research assistent

Tel: +49 (0)211 311 5189

Fax: +49 (0)211 311 3085

Postal address: Institut für Biochemie Geb. 26.42.03

Heinrich-Heine-Universität Düsseldorf

Universitätsstr.1

40225 Düsseldorf

email: biochem@uni-duesseldorf.de

goetz@seqaxp.caltech.edu

Research interests:

Defects in the mitochondrial respiratory chain associated with neurodegenerative diseases
in humans like Morbus Parkinson and idopathic dystonia.

Defects in the mitochondrial respiratory chain associated with neurodegenerative diseases in humans like Morbus Parkinson and idopathic dystonia.

Morbus Parkinson and other neurodegenerative diseases in human appear to be associated with with a male function of the mitochondrial respiratory chain. Serveral enzymes of the mitochondrial respiratory chain show reduced activities in patients suffering from neurodegenerative diseases. However, the malefunction is neither characterized in biochemical terms nor on a moleculare level.

A thorough biochemical investigation is hindered by the minor amounts of protein available as blood or muscle samples from the patients. We are currently immortalizing B-lymphocytes from selected patients by an infection with the Epstein Baar virus. With this material it will be possible to use the biochemical knowledge accumulated in this laboratory with Neurospora and E. coli for an investigation of the defect.

On the genetic level it appears that not a specific mutation is responsible for the defect but a rather unspecific accumulation of mutations in the mtDNA. We are trying to analyse mtDNA from this patients and characterize the defects associated with the mutations by transfer of the mitochondrial DNA to a human mtDNA-less (ro) cell line.

References:

Benecke,R.; Strümper,P.; Weiss,H.; (1993) Electron transfer Complexes I and IV of platelets are abnormal in Parkinson's disease but normal in Parkinson-plus syndromes. Brain 116: 1451-1463.

Beneke,R.; Strümper,P.; Weiss,H.; (1992) Electron Transfer ComplexI Defect in Idiopathic Dystonia. Ann Neurol.32: 683 - 686.

Hofhaus G. and Attardi G.; (1993) Lack of assembly of mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase and loss of enzyme activity in a human cell mutant lacking the mitochondrial ND4 gene product. EMBO J. 12(8) :3043-3048.

Hofhaus G. and Attardi G.; (1995) Efficient Selection And Characterization of Mutants of A Human Cell Line Which Are Defective in Mitochondrial DNA-Encoded Subunits of Respiratory NADH Dehydrogenase. EMBO J. 15(2) 964-974.

Dr. Regina Schneider

Status: Postdoc

Tel: +49 (0)211 311 5156

Fax: +49 (0)211 311 3085

Postal address: Heinrich-Heine-Universitaet Duesseldorf

Institut für Biochemie

Universitaetsstr.1

40225 Duesseldorf

email: regina.schneider@uni-duesseldorf.de

Research interests:

Mitochondrial fatty acid synthesis

The role of mitochondrial respiration in the overproduction of primary metabolites in
Aspergillus niger

THE ROLE OF MITOCHONDRIAL RESPIRATION IN THE OVERPRODUCTION OF PRIMARY METABOLITES IN ASPERGILLUS NIGER

The respiratory chain of filamentous fungi (and plants) contains, apart from the proton-pumping complexes NADH:ubiquinone oxidoreductase (complex I), ubiquinol:cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), so called alternative respiratory enzymes. There are two additional NADH:ubiquinone oxidoreductases (alternative NADH dehydrogenases) concerned with the oxidation of matrix NADH. The proton-pumping NADH:ubiquinone oxidoreductase has a high affinity for NADH, and the non-proton-pumping NADH:ubiquinone oxidoreductase has a low affinity for NADH. We are studying the role of the proton-pumping and alternative NADH:ubiquinone oxidoreductases in the citric acid overproduction by Aspergillus niger. The citric acid overproducing strain B60 was found to lose selectively complex I concomitantly with the onset of the overproduction. To investigate the role of the NADH:ubiquinone oxidoreductases in the catabolism of filamentous fungi, we disrupted in an A. niger wild-type the gene of the (51 kDa) NADH-binding subunit of complex I. A mutant called nuo51 (from NADH:ubiquinone oxidoreductase, 51 kDa subunit) was generated that cannot form a functional complex I. Under growth conditions which lead to uncontrolled catabolic flux through glycolysis, a dramatic catabolic overflow occurred in the mutant nuo51. Intracellular levels of citric acid cycle intermediates rose up to 20 fold normal levels.

References

Prömper, C., Schneider, R. & Weiss, H. (1993) The role of the proton pumping and alternativ respiratoy chain NADH:ubiquinone oxidoreductase in overflow catabolism of Aspergillus niger. Eur. J. Biochem. 216, 223-230.

Schmidt, M., Wallrath, J., Dörner, A. & Weiss, H. (1992). Disturbed assembly of the preassembled membrane arm of NADH:ubiquinone oxidoreductase (complex I) in citric-acid-accumulating Aspergillus niger strain B60. Appl. Micobiol. Biotechnol. 36, 667-672.

Schmidt, M., Friedrich, T., Wallrath, J., Ohnishi, T. & Weiss, H. (1992). Accumulation of the preassembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese-limited grown Neurospora crassa. FEBS Lett. 313, 8-11.

Wallrath, J., Schmidt, M. & Weiss, H. (1991) Concomitant loss of respiratory chain NADH:ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger. Appl. Microbiol. Biotechnol. 36, 76-81.

Wallrath, J., Schmidt, M. & Weiss, H. (1992) Correlation between manganese deficiency, loss of respiratory complex I and citric acid production by Aspergillus niger strain B60. Arch. Microbiol. 158, 435-438.