柴田 直樹

Carboxylesterase (EII') from Arthrobacter sp. KI72 has 88% homology to 6-aminohexanoate-dimer hydrolase (EII) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of EII. To study relationship between Ald-hydrolytic and esterolytic activities, random mutations were introduced into the gene for Hyb-24 (an EII/EII' hybrid with the majority of the sequence deriving for EII' and possessing an EII'-like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fold, and a G181D/D370Y double substitution increased activity ca. 100-fold. On the basis of kinetic studies and the three-dimensional structure of the enzyme, we suggest that binding of Ald is improved by these mutations. D370Y increased esterolytic activity for glycerylbutyrate ca. 30-50-fold, whereas G181D decreased the activity to 30% of the parental enzyme.

Coenzyme B₁₂ or adenosylcobalamin, commonly known as Vitamin B₁₂, is one of the most complicated natural organic compounds involved in heteroatom elimination and carbonskeleton rearrangement reactions. Diol dehydratase is an adenosylcobalamin-dependant enzyme that catalyzes conversion from 1, 2-diol compounds to the corresponding aldehydes. Comparison of the structures of the enzyme of substrate-bound and substrate-free forms reveals the activation mechanism of adenosylcobalamin. Structural studies of chiral substrates imply that this enzyme catalyses each enantiomer with different mechanisms in the final step of the reaction.