Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B 12 -dependent glycerol and diol dehydratases
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ORIGINAL PAPER
Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B12-dependent glycerol and diol dehydratases Takamasa Tobimatsu · Tsuneo Nishiki · Masaya Morimoto · Ryou Miyata · Tetsuo Toraya
Received: 6 August 2008 / Revised: 29 September 2008 / Accepted: 27 October 2008 / Published online: 19 November 2008 © Springer-Verlag 2008
Abstract Coenzyme B12-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since both the N-terminal 20 and 16 amino acid residues of the and subunits, respectively, are indispensable for the low solubility of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the and subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-speciWc Nterminal 34 and 33 amino acid residues of the and subunits, respectively, was not enough to lower the solubility of glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35–60) of the diol dehydratase subunit in addition to the diol dehydratase-speciWc extra-regions of and subunits showed low solubility comparable to diol dehydratase, although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short Nterminal sequences are suYcient to change the solubility of the enzyme.
Communicated by Theo Hansen. T. Tobimatsu · T. Nishiki · M. Morimoto · R. Miyata · T. Toraya (&) Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan e-mail: [email protected]
Keywords Chimeric enzyme · Glycerol dehydratase · Diol dehydratase · Coenzyme B12 · Low solubility Abbreviations AdoCbl Adenosylcobalamin or coenzyme B12 PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction SDS Sodium dodecyl sulfate
Introduction Diol dehydratase (DL-1,2-propanediol hydro-lyase, EC 4.2.1.28) and glycerol dehydratase (glycerol hydro-lyase, EC 4.2.1.30) are isofunctional enzymes, which catalyze the conversion of 1,2-diols, such as 1,2-propanediol, ethanediol and glycerol, to the corresponding aldehydes (Lee and Abeles 1963; Pawelkiewicz and Zagalak 1965; Toraya et al. 1976). These enzymatic reactions are known to proceed by an adenosylcobalamin (AdoCbl)-dependent radical mechanism. They are induced in some genera of Enterobacteriaceae, such as Klebsiella and Citrobacter (Toraya et al. 1978, 1980) and other bacteria (Macis et al. 1998; Toraya 1999), when they are grown anaerobically on 1,2-propanediol or glycerol. They play an essential role in producing electron acceptors for the fermentation of 1,2-diols and glycer
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