Expression, lipoylation and structure determination of recombinant pea H-protein in Escherichia coli

Eur J Biochem. 1996 Feb 15;236(1):27-33. doi: 10.1111/j.1432-1033.1996.00027.x.

Abstract

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.

MeSH terms

  • Amino Acid Oxidoreductases / chemistry*
  • Amino Acid Oxidoreductases / genetics
  • Amino Acid Oxidoreductases / metabolism
  • Apoproteins / chemistry*
  • Apoproteins / genetics
  • Apoproteins / metabolism
  • Base Sequence
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Glycine Decarboxylase Complex
  • Glycine Decarboxylase Complex H-Protein
  • Glycine Dehydrogenase (Decarboxylating)
  • Mass Spectrometry
  • Models, Molecular
  • Molecular Sequence Data
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism
  • Peas / enzymology*
  • Peas / genetics
  • Plant Proteins / chemistry*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Thioctic Acid / chemistry*
  • Thioctic Acid / metabolism
  • Transferases / chemistry*
  • Transferases / genetics
  • Transferases / metabolism

Substances

  • Apoproteins
  • Carrier Proteins
  • Glycine Decarboxylase Complex H-Protein
  • Multienzyme Complexes
  • Plant Proteins
  • Recombinant Proteins
  • glycine cleavage system
  • Thioctic Acid
  • Amino Acid Oxidoreductases
  • Glycine Decarboxylase Complex
  • Glycine Dehydrogenase (Decarboxylating)
  • Transferases