Origine and Molecular Evolutionary Engineering of the Enzyme-Coenzyme Systems

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The stereospecificities for the hydrogen transfer between C-4' of the cofactor and substrate in the transamination catalyzed by various pyridoxal 5'-phosphate (PLP)-dependent enzymes have been studied. The stereospecificities reflect the active-site structures of the enzymes, especially the topographical situation of a coenzyme-substrate Schiff base and a catalytic base for the hydrogen transfer. We found that three types of enzymes catalyzing the reaction stereospecifically on either si-or re-face of the planar intermediate, and alternatively non-stereospecifically on both faces. The classification of PLP-enzymes based on the stereospecificcity for the hydrogen transfer coincident with that based on their primary and three dimensional structures. These suggest that PLP-enzymes have been evolved from at least three different ancestral proteins. These studies lead us the speculation that enzymes requiring coenzymes were created by the combination of the inherently active coenzymes and protein offering the place of the reactions. We attempted to produce a new enzyme by combining a hemin and glutamate recemase showing a significant similarity with mammalian myoglobins in their primary structures.