Research Activity in B₁₂ Area - Karlsruhe Group

To imitate the situation at the active site we are synthesising cobalt-complexes, the so-called cobaloximes, in which the cobalt-carbon bond was intramolecularly fixed by oligomethylene bridge. This ensures a higher stability against irradiation. Then we started the synthesis of such complexes which are additionally equipped with binding sites for artificial substrates. After pioneering work of Jürg Schäffler, Martin Knauer and Berthold Köhler, Rainer Heck has the exciting task to complete the synthesis of a catalytic model.

Rainer Heck

• we use dioxime ligands to mimic (the function of) the corrin ring
• an alkyl chain enables recombination of the Co(II) ion and the methylene radical (formed by thermolysis or photolysis)
• the two cryptands provide a binding site for an artificial substrate like 1,4-diammonium-2,3-butanediol
• as the substrate is not covalently bound to the model the derived ketone (after the rearrangement) could leave the complex making a catalytical turnover possible.
• We proved the stability of the Co-complex by a "small model" (scheme 2)
• The complex was crystallized out of petrol and its structure was confirmed by X-ray analysis.
• The building up of the supramolecular model is currently under investigation.

Scheme 1	Scheme 2

Berthold Köhler spent a year in Newcastle, where he explored the possibility of how stable substitutions at the periphery of alkylcobaloximes can be achieved. The x-ray structure of such a complex has been published with Newcastle researchers as co-authors.¹ Berthold profited from the synthetic skill of the Newcastle colleagues.

Our next synthetic target is being realised by Dr. Eric Kervio. He is transforming vitamin B₁₂ to a semi-synthetic model which, based on similar considerations as illustrated for the cobaloxime model, will hopefully catalyse some of the B₁₂-like rearrangements.

Eric Kervio

The goals of this work are modifications of Vitamine B₁₂ to achieve model catalysts for coenzyme B₁₂ dependent rearrangements. (scheme 3)

This chemical challenge depends on the differenciation and the substitutions of the amide functions as potential reactive centers of the two sides of the corrinoid nucleus. In addition, we had to find an access to original polyfunctionalized structures as synthons for the bridged intramolecular alkylated model, and for the bridged di-crown ether. Studies of activity for these enzyme models by EPR and kinetic measures are planified.

Another subject concerns the role of S-Adenosyl methionine, involved as cofactor in many biosyntheses in plants. It may also undergo homolytic cleavage and produce the 5'-deoxyadenosyl radical (poor man's B₁₂). In exemple, the last step in the biosynthesis of tropane alkaloids is the carbon skeleton rearrangement of littorine to hyoscyamine (cultured hairy roots of Datura stramonium) (scheme 4).

	Scheme 4

It is proposed that S-adenosylmethionine is the source of a 5'-deoxyadenosyl radical which initiates the rearrangement in a similar manner as it does in analogous rearrangements catalyzed by coenzyme B₁₂-dependent enzymes.²

Finally, we are working with several B₁₂-dependent enzymes and have overexpressing E.coli clones of dioldehydratase, glycerol dehydratase, ethanolamine ammonia-lyase, glycerol dehydratase, ethanolamine ammonia-lyase and methylmalonyl-CoA mutase from bovine liver. While the three first enzymes are expressed in large amounts and are active methylmalonyl-CoA mutase could not yet be fold into the active conformation. The structure of ethanolamine ammonia-lyase is being investigated in Graz. Dr. Andreas Abend, at the present postdoc in Madison, and Rainer Nitsche in Karlsruhe investigated the binding mode of coenzyme-B₁₂ in diol and glycerol dehydratases as well as in ethanolamine ammonia-lyase and Rainer will give a short account of these results. He also constructed all possible hybrid enzymes by genetic combination of the subunits of diol and glycerol dehydratases. The hybrids showed interesting kinetics and substrate specifities.

Rainer Nitsche

• High level expression of different B₁₂-enzymes in E. coli, dioldehydratase (S. typhimurium), glyceroldehydratase (C. freundii) and measurement of kinetic behaviour with B₁₂-analogues (collaboration with L. Poppe of TU Budapest)
• Purification of recombinant produced enzymes by IMAC-technology
• Cloning of chimeric dehydratases and investigation of kinetic properties
• Synthesis of ¹⁵N-labelled dimethylbenzimidazole and isolation of ¹⁵N-labelled vitamin B₁₂ (collaboration with E. Stupperich from Ulm)
• EPR measurements with ¹⁵N-labelled coenzyme-B₁₂ with the enzymes glycerol- and dioldehydratases³ and ethanolamine ammonia lyase (collaboration with A. Abend, Madison, Wisconsin), ⁴ribonucleotide reductase (collaboration with J. Stubbe, MIT, Cambridge, USA).⁵

Dagmar Röther

• Subcloning of the genes for ethanolamine ammonia lyase (EAL) in two different vectors, to raise the yield of enzyme and to improve its purification procedure (collaboration for crystallization with Team in Graz)
• Development of a new purification procedure for EAL
• Studies with different adenosylcobalamin-analogues (in cooperation with L. Poppe)

Finally, Torsten Graf is synthesising radioactively labelled inhibitors for the two dehydratases which will hopefully covalently bind to the active site and Uli Weigl is synthesising coenzyme B₁₂ in which the cobalt-bound carbon atom is isotopically labelled. Collaborative efforts with Drs Poppe and Stupperich has been presented by them.

Torsten Graf

It is planned to inhibit the coenzyme-B₁₂-dependent glycerol dehydratase (E.C. 4.2.1.30) with the radioactive labelled inhibitor (4-¹⁴C)-But-3-ene-1,2-diol.

During the enzymatic reaction a radical intermediate emerge from the inhibitor. These radical binds irreversible to the enzyme.

Enzymatic cleavage of the inhibited glycerol dehydratase and isolation of an labelled fragment will allow to identify the binding site of the radical. With this information one can draw conclusions about the course of the enzymatic reaction.

Uli Weigl

• Synthesis of 6'-¹³C-labelled aristeromycin, an analogue of adenosine in which the ribosyl oxygen is replaced by a methylene.
• It's planned to make EPR-measurements with ¹³C-labelled 6'-deoxy-aristeromycil-cobalamin as analog of 5'deoxy-adenosyl-cobalamin.

Birgid Langer

• Purification of an unknown B₁₂-dependant RNA-modifying enzyme which catalyze the reduction of epoxyquenosine (oQ) to quenosine (Q). (scheme 5)

• Isolation of the corresponding gene and sub-cloning for an overexpression vector and kinetical studies.

1. B. Köhler, M. Knauer, W. Clegg, M.R.J. Elsegood, B.T. Golding, J. Rétey, Angew. Chem. 1995, 107, 2530 - 2531; Angew. Chem. Int. Ed. Engl. 1995, 34, 2389 - 2390.
2. S. Ollagnier, E. Kervio, J. Rétey, FEBS Letters 1998, 437, 309-312.
3. A. Abend et al., Angew. Chem. 1998, 110, 643 ; Angew. Chem. Int. Ed. 1998, 37, 625
4. A. Abend et al., J. Biol. Chem. submitted
5. C. C. Lawrence et al., J. Biol. Chem. 1999, 274, 7039