In the group of Prof. M.S. Johnson we have begun modeling the structural framework of the phosphate group binding site in proteins with different folds. As the initial step, we have chosen a vitamin B6-derived coenzyme PLP, which is one of the most versatile cofactors, able to catalyze a wide spectrum of different reactions involved in nitrogen metabolism in all organisms. We have carried out the systematical analysis of PLP containing proteins together with the group of Dr. T. Korpela. Three-dimensional structures of approximately fifty individual PLP-dependent enzymes had been deposited in the  Protein Data Bank at the time of this study. According to the  SCOP database, the proteins belong to five distinct folds. Despite differences in the connectivity of the secondary structure elements that give rise to these five fold types, comparison of the three-dimensional structures of five representative enzymes (one from each fold family): aspartate aminotransferase, beta-subunit of tryptophan synthase, alanine racemase, D-amino acid aminotransferase and glycogen phosphorylase, has revealed extensive structural similarities among their PLP-binding domains  (Denessiouk et al., 1999). The similarities in these enzymes include, among others, two alpha-helices and an adjacent beta-sheet. The phosphate group of PLP is anchored by one of these helices, but the orientations of the pyridoxal rings of PLP are different in the different fold types as are the locations of the active site lysine, to which the pyridoxal ring is bound via the Schiff-base linkage.

The phosphate group binding “cup”
Since the position of the phosphate group is located at the N-terminus of the anchoring alpha-helix in all five families of PLP-dependent proteins, it is of particular interest to know if there exists a common set of atoms with similar chemistry and similar relative orientations that serve to recognize a portion of the pyridoxal phosphate molecule. It was shown that twenty-four structures of PLP-dependent enzymes that represent the five different folds share a common recognition pattern for the phosphate group of their PLP-ligands  (Denesyuk et al., 2002). All atoms that interact with the phosphate group of PLP in these proteins are organized within a two-layer structure so that the first interacting layer contains from five to seven atoms and parallel to this is a second layer containing from three to seven interacting atoms. In order to identify features of the phosphate-binding site common to PLP-dependent enzymes, a simple procedure was described that unambiguously assigns relative positions to all interacting atoms, such that the networks of interactions with the phosphate group for different proteins could be compared. Based on these diagrams for 24 enzyme-cofactor complexes, a detailed comparison of the two-layer structures of PLP-dependent enzymes, with both similar and different folds, was made. A majority of the structurally defined PLP-dependent proteins use the same atom types in analogous “key” positions to bind their PLP-ligands. In some instances, proteins also use water molecules when a key position is unoccupied. A similar two-layer recognition pattern extends to protein recognition of at least one other, non-PLP ligand, glucosamine 6-phosphate. We have named this three-dimensional recognition pattern as the “phosphate-binding cup” .