Atomic Resolution 3D Structure Analysis of Enzymes with a B12 Cofactor

The project aims to obtain atomic resolution 3D structural models for a number of enzymes with a B12 cofactor. This information should eventually lead to an atomic definition of the discrete steps occurring in the active sites of coenzyme B12-dependent enzymes. Specifically, the enzymes glutamate mutase and methyleneglutarate mutase (both from Clostridium Cochlearium) as well as a corrinoid protein from Sporomusa Ovata will be investigated. The prime technique to achieve the structural goals will be macromolecular X-ray crystallography (PX), with specific questions concerning the cofactor`s metal coordination also being addressed by X-ray absorption spectroscopy (XAS). Ancillary techniques, including spectroscopic (UV/VIS, fluorescence and CD spectroscopy) and biocalorimetric (DSC and ITC) techniques, will be employed to address questions of enzyme-enzyme, enzyme-coenzyme, enzyme-substrate and enzyme-inhibitor interactions. Such investigations are planned as preliminaries for subsequent X-ray experiments. The first objective concerns the determination of 3D structures for he native enzyme systems. For glutamate mutase, this will also include the determination of 3D structures of the isolated subunits. Subsequent steps will include studies of crystals with bound inhibitor (at low temperature), with the aim to characterise the reaction intermediate with enzyme-bound adenosyl radical. Further studies will involve synthetically modified coenzymes (e.g. didesoxyadenosyl-cobalamin) to probe enzyme-coenzyme interactions, and to elucidate the mechanism of activation of the coenzyme (Co-C bond cleavage) for the initiation of reactions. As information about active sites emerges, site directed mutagenesis will yield mutants whose structure will reveal the structural and functional role of particular amino acid residues. XAS will be employed to precisely measure the coordination geometry in enzyme-bound B12 in order to assess the relevance of an elongation of the axial cobalt-nitrogen bond for the rate of cobalt-carbon bond cleavage. The research will be carried out in the framework of an interdisciplinary transnational collaboration between 8 European laboratories with complimentary skills, which together form the strongest B12 group in the world. The 8 participants are formally linked by an EU-funded TMR network on "Chemistry and biochemistry of B12 coenzymes and their enzymic partners". Within this network, the applicant`s laboratory is in charge of structure determination of B12-dependent enzymes and protein fragments by X-ray crystallography. The present application requests funding (salaries, consumables and travel expenses) to support one postdoctoral scientist plus two PhD students for three years.

The B12-coenzymes, which have been termed "nature`s most beautiful cofactors", are the most complex, non- polymeric natural products, and they contain the only metal-carbon bond known to exist in biology. They occur as cofactors for about a dozen enzymes, which catalyze chemical reactions that have no precedent in organic chemistry. Adenosylcobalamin-dependent enzymes catalyze radical reactions which are initiated by homolytic cleavage of the cofactors organometallic bond. The way how these enzymes enhance (by about 12 orders of magnitude) the rate of homolytic cleavage of this bond upon binding the substrate, and how they then control the subsequent rearrangement of the high-energy intermediates have fascinated chemists and biologists alike. The aim of the project was to obtain atomic resolution 3D structural models for enzymes with a B12 cofactor and for isolated B12 cofactor analogues. This information should eventually lead to an atomic definition of the discrete steps occurring at the active sites of coenzyme B12-dependent enzymes. The enzyme on which we focussed our interest was glutamate mutase, which catalyzes the equilibration between (S)-glutamate with (2S,3S)-3- methylaspartate. The seminal achievement was the determination of the crystal structure of glutamate mutase from Clostridium cochlearium. This high-resolution structure revealed the mode of binding of a substrate analogue to the enzyme- coenzyme complex and validated the so-called fragmentation-recombination mechanism for the glutamate-mutase catalyzed rearrangement reaction. The 3D structure of the complex between adenosylcobalamin and apo-glutamate mutase revealed the mode of transfer of the intermediately formed adenosyl radical from the B12 cofactor to the substrate. X-ray absorption experiments were showed that the cobalt-carbon bond homolysis is not initiated or significantly enhanced by a enzyme-mediated elongation of the trans-axial cobalt-nitrogen bond, and that the crystallographic observation of such an elongation in the enzyme methylmalonyl-CoA-mutase is most likely an artefact of X-ray crystallography. All these observations contributed significantly the mechanistic understanding of coenzyme B12-dependent reactions. The research involved close collaboration with partners from Germany, Switzerland and Great Britain, who participated in an EU-funded network on "chemistry and biochemistry of B12 coenzymes and their enzymic partners."