Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase
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O R I G I N A L PA P E R
Takamasa Tobimatsu · Hideki Kajiura · Tetsuo Toraya
Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase Received: 3 January 2000 / Revised: 5 May 2000 / Accepted: 8 May 2000 / Published online: 27 June 2000 © Springer-Verlag 2000
Abstract Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase. Key words Adenosylcobalamin · Coenzyme B12 · Glycerol dehydratase · Klebsiella pneumoniae · Diol dehydratase · Klebsiella oxytoca · Reactivating factor · In situ reactivation · Coexpression
T. Tobimatsu · H. Kajiura · T. Toraya (✉) Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan e-mail: [email protected], Tel.: +81-86-2518194, Fax: +81-86-2518264
Introduction Glycerol dehydratase [glycerol hydrolyase (EC 4.2.1.30)] and diol dehydratase [1,2-propanediol hydrolyase (EC 4.2.1.28)] are isofunctional enzymes that catalyze adenosylcobalamin (coenzyme B12)-dependent conversion of glycerol, 1,2-propanediol, and 1,2-ethanediol to the corresponding aldehydes (Toraya 1994). The enzymes are involved in producing electron acceptors for the fermentation of glycerol and 1,2-propanediol via the dihydroxyacetone pathway (Forage and Foster 1982; Forage and Lin 1982; Toraya et al. 1980) and propanediol utilization pathway (Toraya et al. 1979), respectively. They are formed individually or together by genera of the family Enterobacteriaceae, such as Klebsiella and Citrobacter, and other bacteria when the cells are grown anaerobically in a medium containing glycerol and/or 1,2-propanedi
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