Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisalpha143. Mutant enzymes in which Hisalpha143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Halpha143Q mutant was 34% as active as the wild-type enzyme. Halpha143A and Halpha143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue alpha143 is important not only for catalysis but also for protecting radical intermediates. Halpha143E and Halpha143K that did not exist as (alphabetagamma) 2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Halpha143Q and partially rate-determining for Halpha143A. The preference for substrate enantiomers was reversed by the Halpha143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Halpha143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5'-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Halpha143Q underwent irreversible inactivation by O 2 in the absence of substrate at a much lower rate than the wild type.

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.

Henry's law constants and infinite dilution activity coefficients of propane, propene, butane, 2-methylpropane, 1-butene, 2-methylpropene, trans-2-butene, cis-2-butene, 1,3-butadiene, dimethyl ether, chloroethane, and 1,1-difluoroethane in benzene, toluene, o-xylene, m-xylene, p-xylene, and styrene in the temperature range of (250 to 330) K were measured by a gas stripping method, and partial molar excess enthalpies and entropies were evaluated from the activity coefficients. A rigorous formula for evaluating the Henry's law constants from the gas stripping measurements was used for the data reduction of these highly volatile mixtures. The estimated uncertainties are about 2 % for the Henry's law constants and 3 % for the infinite dilution activity coefficients. The Henry's law constants followed the order of increasing Henry's law constant with decreases in the normal boiling point temperature of the liquefied gas except polar gases. In general, the partial molar excess enthalpies and entropies of gases in the aromatics increase with decreases of the polarities of the gases and increasing molecular size of the gases.

Summary Adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase are isofunctional enzymes that catalyze the dehydration of 1,2-diols to the corresponding aldehydes. Although they bear different metabolic roles, both enzymes consist of three different subunits and possess a common ( ␣␤␥ ) 2 structure. To elucidate the roles of each subunit, we constructed expression plasmids for the hybrid dehydratases between diol dehydratase of Klebsiella oxytoca and glycerol dehydratase of Klebsiella pneumoniae in all the combinations of subunits by gene engineering techniques. All of the hybrid enzymes were produced in Escherichia coli at high levels, but only two hybrid enzymes consisting of the ␣ subunit from glycerol dehydratase and the ␤ subunits from diol dehydratase showed high activity. The substrate specificity, the susceptibility to inactivation by glycerol, and the monovalent cation specificity of the wild type and hybrid enzymes were primarily determined by the origin of their ␣ subunits. Key Words coenzyme B 12 , adenosylcobalamin, diol dehydratase, glycerol dehydratase, hybrid enzymes Diol dehydratase ( DL -1,2-propanediol hydro-lyase, EC 4.2.1.28) and glycerol dehydratase (EC 4.2.1.30) are isofunctional enzymes that catalyze the AdoCbl 1 -dependent conversion of 1,2-diols to the corresponding deoxy aldehydes ( 1-3 ). They consist of the three different subunits, ␣ ( Mr 60,000-61,000), ␤ ( Mr 21,000-24,000), and ␥ ( Mr 16,000-19,000), and dissociate into two dissimilar protein components in the absence of substrate ( 4, 5 ). Large and small components correspond to the ␣ 2 ␥ 2 complex and the ␤ subunit, respectively ( 6, 7 ). Their catalytic properties are similar, but they are different in the binding affinity for AdoCbl ( 8,9 ), substrate specificity ( 1-3, 10, 11 ), susceptibility to suicide inactivation by glycerol ( 2, 9, 10 ), monovalent cation specificity ( 1,11,12 ), and immunochemical reactivity toward anti-diol dehydratase antiserum ( 11 ) (for review, see Refs. 13-15 )). In order to reveal their similarities and differences on a molecular level, we cloned, sequenced, and expressed the diol dehydratase genes of Klebsiella oxytoca ( 16 ) and K lebsiella pneumoniae ( 17 ) and the glycerol dehydratase genes of K. pneumoniae ( 18 ). The glycerol dehydratase genes of Citrobacter freundii ( 19 ) and Clostridium pasteurianum ( 20 ) and the diol dehydratase genes of Salmonella typhimurium ( 21 ) and Lactobacillus collinoides ( 22 ) have also been cloned by other investigators. Comparison of the deduced amino acid sequences of the corresponding subunits between diol and glycerol dehydratases indicated that the ␣ subunit is most highly conserved among the subunits of the enzymes (identity, 71%), whereas the homologies of ␤ and ␥ subunits are lower (identities, 58% and 54%, respectively) ( Fig. 1) ( 18 ). The N-terminal 32 and 37 amino acid residues of the ␤ and ␥ subunits, respectively, of diol dehydratase are lacking in the corresponding subunits of glycerol dehydratase (16)(17)(18). These regions ...