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

The glycine decarboxylase complex consists of four different component enzy mes (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 prostheti c group (lipoic acid) interacts successively with the three other component s of the complex and undergoes a cycle of reductive methylamination, methyl amine transfer and electron transfer. With the aim to understand the intera ction between the H-protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms o f the H-protein. In the present study, we have crystallized the H-protein i n its reduced state and the L-protein (lipoamide dehydrogenase or dihydroli poamide dehydrogenase). The L-protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were o btained from the refolded L-protein and the structure has been determined b y X-ray crystallography. This first crystal structure of a plant dihydrolip oamide dehydrogenase is similar to other known dihydrolipoamide dehydrogena se structures. The crystal structure of the H-protein in its reduced form h as 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 contra st with methylaminated H-protein where the loaded lipoamide arm was locked into a cavity of the protein, the reduced lipoamide arm appeared freely exp osed to the solvent. Such a freedom is required to allow its targeting insi de 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 hypot hesis is supported by biochemical data [Neuburger, M., Polidori, A.M., Piet re, E., Faure, M., Jourdain, A., Bourguignon, J., Pucci, B. & Douce, R. (20 00) Eur. J. Biochem. 267, 2882-2889] and by small angle X-ray scattering ex periments reported herein.