Cobalamin (vitamin B12) biosynthesis--cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine-dependent cobalt-precorrin-4 transmethylase CbiF

Eur J Biochem. 1998 Jun 1;254(2):341-6. doi: 10.1046/j.1432-1327.1998.2540341.x.

Abstract

The Bacillus megaterium cbiF, encoding the cobalt-precorrin-4 S-adenosyl-L-methionine-dependent transmethylase of the anaerobic cobalamin biosynthetic pathway, has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli. The protein was purified to homogeneity by a combination of metal chelate chromatography and high-resolution anion-exchange chromatography. The protein migrated with a subunit mass of 31 kDa by SDS/PAGE and with a molecular mass of 62 kDa by analytical gel filtration, suggesting that the native recombinant protein is a homodimer. The His-tagged protein was physiologically active as it was able to complement a Salmonella typhimurium cbiF mutant. However, the protein did not bind S-adenosyl-L-methionine with the same avidity as observed with other corrin biosynthetic transmethylases. A crystallisation screen of the purified protein led to the identification of two discrete crystal forms. One of these forms has been characterised and a full data set collected.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacillus megaterium / enzymology*
  • Bacillus megaterium / genetics*
  • Base Sequence
  • Cloning, Molecular
  • Cobalt / metabolism
  • Crystallization
  • DNA Primers / genetics
  • Dimerization
  • Escherichia coli Proteins*
  • Gene Expression
  • Genes, Bacterial
  • Methyltransferases / genetics*
  • Methyltransferases / isolation & purification
  • Methyltransferases / metabolism
  • Molecular Weight
  • Protein Conformation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • S-Adenosylmethionine / metabolism
  • Vitamin B 12 / biosynthesis*

Substances

  • DNA Primers
  • Escherichia coli Proteins
  • Recombinant Proteins
  • Cobalt
  • S-Adenosylmethionine
  • Methyltransferases
  • precorrin-3 11-methyltransferase
  • precorrin-4 methyltransferase, E coli
  • Vitamin B 12