2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes

Eur J Biochem. 2001 Jun;268(12):3577-86. doi: 10.1046/j.1432-1327.2001.02262.x.


In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources ( approximately 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mM pyruvate, 150 mM succinate and 10 microM PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspergillus nidulans / enzymology*
  • Carbon-Carbon Lyases / antagonists & inhibitors
  • Carbon-Carbon Lyases / chemistry
  • Carbon-Carbon Lyases / genetics
  • Carbon-Carbon Lyases / isolation & purification*
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology*
  • Molecular Sequence Data
  • Sequence Homology, Amino Acid
  • Stereoisomerism


  • Carbon-Carbon Lyases
  • methylisocitrate lyase