Structure and molecular interaction of B12-binding proteins

Nucleotides and nucleotide-binding proteins have central roles in life processes. By binding and activating nucleotidic cofactors, enzymes make use of the controlled and metabolically important reactivity of a variety of mono-, di- and trinucleotides. To the latter belong the ubiquitous vitamin B12 derivatives, such as the corrinoid di- and trinucleotides methylcobalamin and coenzyme B12, that have unique organometallic functions in a broad range of forms of life. The present project is planned to deal with some enzymes, whose functions are based on their ability to bind and activate B12-cofactors. Some of these enzymes have been recognized recently to belong to the larger group of the nucleotide-binding proteins. Part of the proposed work is concerned with the problem of specific recognition, binding and activation of the organometallic trinucleotide coenzyme B12 by the B12-binding subunit of a coenzmye B12-dependent bacterial glutamate mutase. The structure of the B12-binding protein and the structural elements that lead to specific binding of the B12-cofactor are to be investigated in solution by spectroscopic means. These studies will be extended to structural investigations of B12-binding by some newly developed monoclonal B12-antibodies, whose related modes of interaction with coenzyme B12 and some of its organometallic analogues will be analyzed by spectroscopic, means. Connected with these studies, the mode of binding of vitamin B12- derivatives to the human B12-binding protein "intrinsic factor" will also be investigated structurally. The project is planned to help unravel the structural basis and the motifs of recognition of some proteins that bind and activate the unusual organometallic di- and trinucleotidic B12-coenzymes, of importance for human and other forms of life.

Vitamin B12 and its derivatives are vitamins for humans and have central roles in almost all forms of life. They provide the basis for essential life processes, which depend upon a series of unique organometallic reactions provided by B12. By binding and activating the B12-cofactors, enzymes make use of the metabolically important reactivity of these B12-derivatives. Some of these enzymes have been recognized recently to belong to the larger group of the nucleotide-binding proteins. The project was planned to deal with the problem of how coenzyme B12 and related B12-derivatives are recognized, bound and activated by binding to proteins whose functions depend on their ability to use the unique B12-derivatives as organometallic cofactors. The structural elements that lead to specific binding of such B12-cofactors by a naturally occurring B12-binding protein were thus investigated in great detail by spectroscopic means. The work carried out here concerned the properties in solution of the B12-binding domain of the coenzyme B12-dependent glutamate mutase, an enzyme catalyzing a complex carbon skeleton rearrangement. Our experiments showed how the nucleotide moiety of B12 was crucial for recognition of coenzyme B12 by B12-binding proteins and provided the basis for a proposal for the binding mechanism. Monoclonal antibodies could be developed, with a specific capacity to bind coenzyme B12 and related organometallic B12-derivatives. Structural investigations of these artificial B12-binding proteins by modern spectroscopic means were carried out and gave detailed insights into the modes of interaction with coenzyme B12 and some of its organometallic analogues. In the same context, as these studies, experiments on the mode of binding of vitamin B12-derivatives to natural human B12-binding proteins (such as "intrinsic factor") were taken up and their capacity to specifically recognize and bind B12-derivatives was investigated structurally. These experiments assigned the B12-nucleotide moiety a crucial role in the ability of such natural and essential B12- binding proteins to recognize B12. In addition to the protein-based B12-dependent enzymes, metabolically important direct interactions between B12- cofactors and RNA were recently discovered. A third line of experiments thus targeted the interaction of B12 with DNA and RNA. Covalent conjugates of DNA with B12 were designed, and a method for their synthesis was worked out, to reveal some of the consequences of such direct interactions with B12-cofactors. The project thus helped to unravel the structural basis of bio-macromolecules that bind and activate the unusual organometallic B12-coenzymes.