Cobalamin (vitamin B12) biosynthesis--cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine-dependent cobalt-precorrin-4 transmethylase CbiF.
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.
Raux
E
E
Department of Molecular Genetics, Institute of Ophthalmology, University College London, UK.
Schubert
H L
HL
Woodcock
S C
SC
Wilson
K S
KS
Warren
M J
MJ
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003035:Cobalt / Q000378:metabolism; D003460:Crystallization; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D014805:Vitamin B 12 / Q000096:biosynthesis*
D017931:DNA Primers; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D003035:Cobalt; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C078454:precorrin-3 11-methyltransferase; C094568:precorrin-4 methyltransferase, E coli; D014805:Vitamin B 12