Structural and functional characterization of H protein mutants of the glycine decarboxylase complex.
The mitochondrial glycine decarboxylase complex (GDC) consists of four component enzymes (P, H, T, and L proteins) involved in the breakdown of glycine. In order to investigate structural interactions involved in the stabilization of the methylamine-loaded H protein (a transient species in the GDC reaction), we designed several mutants of H apoprotein. Structural analysis of the wild-type and mutants of H apoprotein emphasized the necessity to carefully assess, by biophysical techniques, the correct folding of mutated proteins prior to investigate their biochemical properties. The correctly folded wild-type and mutants of H apoprotein were in vitro lipoylated and then characterized in the context of GDC reaction by studying the reconstituted complex and partial reactions. We showed that Val(62) and Ala(64), 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 recognition of the binding site of lipoic acid by lipoyl ligase. The biochemical results obtained with the HE14A mutant of H protein pointed out the major role of the Glu(14) amino acid residue in the GDC catalysis and highlighted the importance of the ionic and hydrogen bounds in the hydrophobic cleft of H protein for the stabilization of the methylamine-loaded lipoyl arm.
Gueguen
V
V
Laboratoire de Physiologie Cellulaire Végétale, URA 576, Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, Département de Biologie Moléculaire et Structurale, 328097 Grenoble cedex 1, France.
Macherel
D
D
Neuburger
M
M
Pierre
C S
CS
Jaquinod
M
M
Gans
P
P
Douce
R
R
Bourguignon
J
J
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D050959:Glycine Decarboxylase Complex; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D009682:Magnetic Resonance Spectroscopy; D016296:Mutagenesis; D018532:Pisum sativum / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008063:Thioctic Acid / Q000378:metabolism
D001059:Apoproteins; D017931:DNA Primers; D050961:Glycine Decarboxylase Complex H-Protein; D011994:Recombinant Proteins; D008063:Thioctic Acid; D000594:Amino Acid Oxidoreductases; D050959:Glycine Decarboxylase Complex; D050841:Glycine Dehydrogenase (Decarboxylating)