Expression, lipoylation and structure determination of recombinant pea H-protein in Escherichia coli.
A synthetic gene encoding the entire mature H protein of the glycine decarboxylase complex from pea (Pisum sativum L.) was constructed and expressed in Escherichia coli. The recombinant H protein, which after the induction period constituted more than half of the E. coli protein, was found in a soluble form. Activity measurements and mass-spectrometry analysis of the purified protein showed that, in the absence or presence of 5[3-(1,2)-dithiolanyl]pentanoic acid (lipoic acid) in the culture medium, recombinant H protein could be produced as the unlipoylated apoform or as the lipoylated form, respectively. Addition of chloramphenicol to the culture medium after induction increased the proportion of lipoylated H protein. High rates of lipoylation of the H apoprotein were measured in vivo and in vitro, revealing that the recombinant pea H protein was an excellent substrate for the E. coli lipoyl-ligase. The three-dimensional structure of the recombinant H apoprotein was determined at a 0.25-nm resolution. It was almost identical to the structure of the native pea leaf enzyme, which indicates that the recombinant protein folds properly in E. coli and that the lipoyl-ligase recognizes a three-dimensional structure in order to add lipoic acid to its specific lysine residue. It is postulated that the high level of expression and lipoylation of recombinant H protein may be due to the protein retaining the structure of the original enzyme.
Macherel
D
D
Laboratoire de Physiologie Cellulaire Végétale, URA CNRS no. 576, Départment de Biologie et Moléculaire Structurale, CEA/Grenoble, France.
Bourguignon
J
J
Forest
E
E
Faure
M
M
Cohen-Addad
C
C
Douce
R
R
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D050959:Glycine Decarboxylase Complex; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D008063:Thioctic Acid / Q000737:chemistry* / Q000378:metabolism; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001059:Apoproteins; D002352:Carrier Proteins; D050961:Glycine Decarboxylase Complex H-Protein; D009097:Multienzyme Complexes; D010940:Plant Proteins; D011994:Recombinant Proteins; C064525:glycine cleavage system; D008063:Thioctic Acid; D000594:Amino Acid Oxidoreductases; D050959:Glycine Decarboxylase Complex; D050841:Glycine Dehydrogenase (Decarboxylating); D014166:Transferases