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Holopropanediol dehydratase (EC 4.2.1.28) was resolved completely into the apoenzyme and corrinoid(s) upon gel filtration on a column of Sephadex G-25 in the absence of potassium ion. The apoprotein obtained by this resolution procedure could be reconstituted into the catalytically active holoenzyme by the incubation at 37' with added B12 coenzyme in the presence of potassium ion. The recovery of dehydratase activity was nearly quantitative. The major corrinoid recovered was identified as 5 '-deoxyadenosylcobalamin. Of inactive complexes between the apoenzyme and irreversible cobamide inhibitors of propanediol dehydratase, cyanocobalamin-and methylcobalamin-apoenzyme complexes were also mostly resolved upon the gel filtration in the absence of both potassium ion and the substrate, yielding the apoenzyme which was reconstitutable into the active holoenzyme. Hydroxocobalamin-apoenzyme complex, however, was hardly resolvable under the same conditions. In the presence of both potassium ion and the substrate, gel filtration of the holo-I t has been generally considered that vitamin B12 coenzyme or its analogs bind to the apoprotein of propanediol dehydratase (DL-1 ,Zpropanediol hydro-lyase, EC 4.2.1.28) or to those of many other Ble coenzyme-dependent enzymes almost irreversibly and the resolution of B12 coenzyme-apoenzyme complex (holoenzyme) or coenzyme analog-apoenzyme complex into the active apoprotein and corresponding cobalamin is usually accompanied by a significant loss of the enzyme activity. Charcoal treatment or exhaustive dialysis was found not to be effective. An acid ammonium sulfate resolution procedure, which was effectively used for mammalian methylmalonyl-CoA mutase (Lengyel et al., 1960) and ethanolamine deaminase (Kaplan and Stadtman, 1968b), led to a marked decrease in the propanediol dehydratase activity (T. Toraya, S. Shimizu, and S. Fukui, 1969, unpublished results).During the course of an investigation on factors necessary for binding of B12 coenzyme or its analogs to the apoprotein of propanediol dehydratase, our attention was drawn to the function of a monovalent cation which was required as a cofactor by this enzyme. Although it has previouslycbeen reported that propanediol dehydratase and many other coenzyme BIZ dependent enzymes show an absolute requirement for a monovalent cation, such as potassium ion, for their catalytic activitieslSmiley and Sobolov. Receiued March 15, 1971. A preliminary report of portions of this work has been published (Toraya er al., 1970).To whom correspondence should be addressed. enzyme or coenzyme analog-apoenzyme complexes resulted in negligible resolution. When gel filtration of the holoenzyme was carried out in the presence of potassium ion only, the holoenzyme was found to be resolved according to the kinetics of first-order reaction. These experiments indicate that potassium ion plays an essential role in the binding of the apoenzyme with coenzyme B I~ or its analogs except for hydroxocobalamin. Only hydroxocobalamin could bind to the apoenzyme even in t...

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme-BIZ-dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme-BI2-dependent diol dehydratase in a 1,2-propanediolcontaining medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneurnoniae ( A . aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent-cation-selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2-propanediol as a substrate, whereas diol dehydratase preferred 1,2-propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double-diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneurnoniue ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.

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Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2diols, to acids and alcohols. The presence of activities of coenzyme B12-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera ofEnterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B12-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B12 was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.