The TMR B12 Network

A Collaborative Project Across Europe

Network Participants:
Newcastle (United Kingdom), Marburg (Germany), Karlsruhe (Germany), Bern (Switzerland), Innsbruck (Austria), Graz (Austria), Ulm (Germany), Budapest (Hungary)

Meeting Announcements:

The 5^th European Symposium on
Vitamin B₁₂ and B₁₂-Proteins
will be held in Marburg
10^th - 15^th September, 2000

Also, a related meeting:

Radicals in Enzymatic Catalysis
will be held in Marburg
12^th - 14^th April, 2000

Previous Meetings:

The last network meeting was held in Graz on November 27th -28th with a
crystallography workshop November 29th - December 1st 1999.

The Budapest meeting was held 18th-20th of June 1999.

The B12 Toolbox was held in Marburg the 5th-9th of May 1999.

Contact Addresses

Objectives of the Network:

The network aims to define with atomic precision the discrete steps occurring at the active sites of enzymes dependent on coenzyme forms of vitamin B₁₂. These enzymes catalyse an array of reactions important for both prokaryotic and eukaryotic organisms, including humans. The research programme will enable sufficient of the individual enzymes to be isolated for structural analyses by X-ray and spectroscopic methods. The hypothesis ('fragmentation-recombination mechanism') that emerged from a previous programme requires verification. To this end, electron paramagnetic resonance spectroscopy will be used to probe the nature of protein-bound, intermediate free radicals and to search for radical centres that may be located on the protein. In parallel with the enzymic studies, modified and isotopically labelled coenzymes will be used to probe enzyme-coenzyme interactions, and especially the mode of activation of the coenzyme for the initiation of reactions: homolytic Co-C bond cleavage for the coenzyme called adenosylcobalamin, heterolytic cleavage for the coenzyme methylcobalamin. Model studies will attempt to replicate all of the main features of the reaction pathways accomplished by the B12-dependent enzymes.

Results and Achievements:

Scientific Highlights

The crystal structure of the enzyme glutamate mutase from Clostridium cochlearium has been elucidated (Graz and Marburg) and shows clearly the mode of substrate binding.
The solution structure of component S of glutamate mutase from Clostridium cochlearium has been determined by NMR (Innsbruck and Marburg).
The substrate-derived radical (4-glutamyl, i.e. the key intermediate) in the glutamate mutase reaction has been characterised by EPR (Marburg and Newcastle).
New insights into reaction mechanisms based on ab initio molecular orbital calculations of reaction pathways have been gained by an international co-operation with Canberra (Newcastle).
Efficient model systems for methyltransferases have been developed, e.g. activation of methanol using cob(I)alamin (Bern).
Routine gene technological methods for obtaining recombinant coenzyme B₁₂-dependent enzymes have been developed (Karlsruhe, Innsbruck, Marburg and Ulm).

Keywords:

Vitamin; enzyme; coenzyme; rearrangement; radical.