Purification and properties of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanobacterium thermoautotrophicum

Eur J Biochem. 1993 Apr 1;213(1):537-45. doi: 10.1111/j.1432-1033.1993.tb17792.x.

Abstract

N5-Methyltetrahydromethanopterin:coenzyme M meth-yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy-conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purified 30-fold to apparent homogeneity. The purified enzyme had an apparent molecular mass of 670 kDa and was composed of seven different polypeptides of 34 kDa, 28 kDa, 24 kDa, 23 kDa, 21 kDa, 13 kDa, and 12 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native 670-kDa enzyme was found to contain 7.6 mol 5-hydroxybenzimidazolyl cobamide/mol, 37 mol non-heme iron/mol and 34 mol acid-labile sulfur/mol. Cobalt analyses after sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that the corrinoid was bound to the 23-kDa polypeptide. The apparent molecular masses of the polypeptides given above were determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without boiling the samples prior to analysis. When the samples were boiled, as is usually done, the 23-kDa polypeptide changed its apparent molecular mass to 33 kDa and the 21-kDa, 24-kDa, and 28-kDa polypeptides formed aggregates. The specific activity (apparent Vmax) of the purified methyltransferase preparation was 11.6 mumol.min-1.mg protein-1. The apparent Km for N5-methyltetrahydromethanopterin was 260 microM and that for coenzyme M was 60 microM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5-2-fold.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalysis
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Corrinoids
  • Electrophoresis, Polyacrylamide Gel
  • Membrane Proteins / chemistry
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism
  • Metalloproteins / chemistry
  • Metalloproteins / isolation & purification*
  • Metalloproteins / metabolism
  • Methanobacterium / enzymology*
  • Methyltransferases / chemistry
  • Methyltransferases / isolation & purification*
  • Methyltransferases / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Nonheme Iron Proteins
  • Vitamin B 12 / metabolism

Substances

  • Corrinoids
  • Membrane Proteins
  • Metalloproteins
  • Nonheme Iron Proteins
  • Methyltransferases
  • tetrahydromethanopterin S-methyltransferase
  • Vitamin B 12

Associated data

  • GENBANK/P80183
  • GENBANK/P80184
  • GENBANK/P80185
  • GENBANK/P80186
  • GENBANK/P80187
  • PIR/P80183
  • PIR/P80184
  • PIR/P80185
  • PIR/P80186
  • PIR/P80187
  • SWISSPROT/P80183
  • SWISSPROT/P80184
  • SWISSPROT/P80185
  • SWISSPROT/P80186
  • SWISSPROT/P80187