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Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).

Proceedings of the National Academy of Sciences of the United States of America (2013-08-08)
Simon J Moore, Andrew D Lawrence, Rebekka Biedendieck, Evelyne Deery, Stefanie Frank, Mark J Howard, Stephen E J Rigby, Martin J Warren
ABSTRACT

It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Vitamin B12, ≥98%
Sigma-Aldrich
Vitamin B12, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥98%
Supelco
Cyanocobalamin (B12), analytical standard
Sigma-Aldrich
Cyanocobalamin, meets USP testing specifications
Sigma-Aldrich
Cyanocobalamin, tested according to Ph. Eur.
Supelco
Cyanocobalamin (Vitamin B12) solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®
Supelco
Cyanocobalamin, pharmaceutical secondary standard, certified reference material