Raux, E. and Warren, M.J. (1998) Cobalamin (vitamin B-12) biosynthesis - Cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine-dependent cobalt-precorrin-4 transmethylase CbiF. European Journal of Biochemistry, 254 (2). pp. 341-346. ISSN 0014-2956 .
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| Official URL http://dx.doi.org/10.1111/j.1742-4658.2009.07300.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.
| Item Type: | Article |
|---|---|
| Uncontrolled keywords: | vitamin b-12 cbif cobalt-preconin-4 bacillus megaterium s-adenosyl-l-methionine uroporphyrinogen-iii methyltransferase pseudomonas-denitrificans salmonella-typhimurium sequence-analysis genes purification synthase intermediate precorrin-2 acid |
| Subjects: | Q Science |
| Divisions: | Faculties > Science Technology and Medical Studies > School of Biosciences |
| Depositing User: | Martin Warren |
| Date Deposited: | 03 Oct 2009 11:42 |
| Last Modified: | 14 Jan 2010 14:42 |
| Resource URI: | http://kar.kent.ac.uk/id/eprint/11083 (The current URI for this page, for reference purposes) |
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