You are here : Home > LPCV > Structural and functional studies of the protein H of the mitochondrial glycine decarboxylase complex and characterization of the biosynthesis of one of its cofactors: The lipoic acid

Virginie Gueguen

Structural and functional studies of the protein H of the mitochondrial glycine decarboxylase complex and characterization of the biosynthesis of one of its cofactors: The lipoic acid

Published on 22 February 2000

Thesis presented February 22, 2000

H protein, a lipoate-containing protein of the mitochondrial glycine decarboxylase complex (GDC), plays a central role in the oxidative decarboxylation and deamination of glycine. It interacts sequentially with the three others components of the complex, P, T and L proteins, through its lipoamide arm covalently bound to a specific lysine residue (K63 in Pisum sativum). When glycine is decarboxylated by P protein, a pyridoxal-phosphate containing protein, the remaining methylamine group is bound to the lipoic acid, which moves in a hydrophobic cleft at the surface of H protein. Using site-directed mutagenesis of E14, S12 and I27 residues from the cavity, we investigated structural interactions that were likely to stabilize the methylamine-lipoyl arm within the hydrophobic cleft. The recombinant mutant and wild-type H-apoproteins were analyzed by several biophysical techniques and then biochemically characterized in the context of GDC reaction, by studying the reconstituted complex and partial reactions. Structural analysis demonstrated that a single mutation could upset the structure of the proteins, resulting in predominantly unfolded proteins. Functional study of the correctly folded mutant H-apoprotein demonstrated that the crucial role of interactions of E14 with the NH3+ group of methylamine and carboxyl​ group of S12 in the stabilization of the methylamine loaded H-protein, a transient species in GDC reactions. Besides, the structural and functional similarity between lipoate proteins and biotin-dependent proteins prompted us to construct a double mutant in which the VKA motif of the lipoyl domain was replaced by the MKM motif of the biotinyl domain. We showed that V62 and A64 surrounding the lipoyl-lysine play an important role in the molecular events that govern the reaction between P and H protein, but do not intervene in the lipoylation mechanism.
In a second part, we demonstrated lipoic acid biosynthesis in plant mitochondria. The pathway could be analogous to FAS II system from bacteria and chloroplasts, leading to C4 to C18 acyls-ACP. Malonic acid is, specifically to mitochondria, the precursor of lipoic acid and long chain fatty acids synthesis. About ten of independent enzymes could intervene to elongate acyls-ACP. We started biochemical characterization of the enzymes which initiate the pathway and manipulate malonic acid. Kinetic parameters of malonyl-CoA synthetase (MCS) which activate malonic acid in malonyl-CoA were determinated. Two more enzymatic activities were characterized, the malonyl-CoA:ACP transacylase (MC:AT), which rapidly catalyze the conversion of malonyl-CoA in malonyl-ACP, and the malonyl-ACP synthetase (MAS) which allows malonyl-ACP synthesis directly from malonic acid. Elongation of malonyl-ACP unit such synthetized is assured by condensing enzymes and leads to two main acyls-ACP products: octanoyl-ACP and hexadecanoyl-ACP. The first one allows lipoic acid biosynthesis thanks to sulfur insertion by an unknown donor and the second one leads to long chain fatty acids. We showed that H-apoprotein could be octanoylated or lipoylated by matrix extract from malonic acid. Thus, matrix ows all the enzymatic equipment to assure the posttranslationnal modification of H-apoprotein, from lipoic acid biosynthesis from malonic acid to its binding by a lipoyl ligase enzyme.

H protein, Mitochondrial glycine decarboxylase complex, GDC, lipoate, H apoprotein