title: Interaction between the lipoamide-containing H-protein and the lipoamide dehydrogenase (L-protein) of the glycine decarboxylase multienzyme system 2. Crystal structures of H- and L-proteins.
abstract: The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions as its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. With the aim to understand the interaction between the H-protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms of the H-protein. In the present study, we have crystallized the H-protein in its reduced state and the L-protein (lipoamide dehydrogenase or dihydrolipoamide dehydrogenase). The L-protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were obtained from the refolded L-protein and the structure has been determined by X-ray crystallography. This first crystal structure of a plant dihydrolipoamide dehydrogenase is similar to other known dihydrolipoamide dehydrogenase structures. The crystal structure of the H-protein in its reduced form has been determined and compared to the structure of the other forms of the protein. It is isomorphous to the structure of the oxidized form. In contrast with methylaminated H-protein where the loaded lipoamide arm was locked into a cavity of the protein, the reduced lipoamide arm appeared freely exposed to the solvent. Such a freedom is required to allow its targeting inside the hollow active site of L-protein. Our results strongly suggest that a direct interaction between the H- and L-proteins is not necessary for the reoxidation of the reduced lipoamide arm bound to the H-protein. This hypothesis is supported by biochemical data [Neuburger, M., Polidori, A.M., Piètre, E., Faure, M., Jourdain, A., Bourguignon, J., Pucci, B. & Douce, R. (2000) Eur. J. Biochem. 267, 2882-2889] and by small angle X-ray scattering experiments reported herein.
authors.affiliation: Institut de Biologie Structurale Jean-Pierre Ebel, CEA/CNRS/Université Joseph Fourier, Grenoble, France.
authors.forename: M
authors.initials: M
authors.lastname: Faure
authors.forename: J
authors.initials: J
authors.lastname: Bourguignon
authors.forename: M
authors.initials: M
authors.lastname: Neuburger
authors.forename: D
authors.initials: D
authors.lastname: MacHerel
authors.forename: L
authors.initials: L
authors.lastname: Sieker
authors.forename: R
authors.initials: R
authors.lastname: Ober
authors.forename: R
authors.initials: R
authors.lastname: Kahn
authors.forename: C
authors.initials: C
authors.lastname: Cohen-Addad
authors.forename: R
authors.initials: R
authors.lastname: Douce
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D050959:Glycine Decarboxylase Complex; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D050961:Glycine Decarboxylase Complex H-Protein; D011994:Recombinant Proteins; C007409:dihydrolipoamide; D008063:Thioctic Acid; D000594:Amino Acid Oxidoreductases; D050959:Glycine Decarboxylase Complex; D050841:Glycine Dehydrogenase (Decarboxylating); D008058:Dihydrolipoamide Dehydrogenase