Studies on the Structure and the Mechanism of Radical Enzymes

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Electron paramagnetic resonance (EPR) measurements were conducted to elucidate the mode of binding of cobalamin to diol dehydratase. It was demonstrated that this enzyme binds cobalamin in the ゛base-on″mode, and that the bulkiness of the lower axial base is important for protection of reactive radical intermediates. Two forms of crystals of the diol dehydratase-cyanocobalamin complex were obtained, which diffracted up to 2.2 and 3.0 A resolution. The crystal structure of the complex revealed that cobalamin is bound to the enzyme in the base-on mode. The active site exists inside the TIM barrel which may protect reactive radical intermediates from side reactions. The two hydroxyl groups of the substrate coordinate directly to KィイD1+ィエD1 in the active site. Density functional theory computations indicated that the hydroxyl group migration from C-2 to C-1 proceeds by a concerted pathway through a cyclic transition state. KィイD1+ィエD1 seems to be important not only in stabilizing the transition state but also in labilizing the Co-C bond indirectly by increasing the substrate binding energy.The products of putative reactivating factor genes were purified and confirmed in vitro to function as a reactivating factor for glycerol-inactivated and oィイD22ィエD2-inactivated holoenzymes. The mechanism of action of the diol dehydratase-reactivating factor was investigated. It was demonstrated that ATP- and ADP -forms of this factor are low and high affinity forms for diol dehydratase, and that the modified coenzyme in the inactivation is released from the enzyme and, as a result, substituted by intact coenzyme, reconstituting catalytically active holoenzyme. This factor is thus considered as a sort of molecular chaperone. The two genes in proximity to the glycerol dehydratase genes were identified as the genes for a glycerol dehydratase-reactivating factor. Their amino acid sequences showed high homology with the diol dehydratase-reactivating factor.