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Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B12-dependent glycerol and diol dehydratases

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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 β and γ 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 β and γ subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific N-terminal 34 and 33 amino acid residues of the β and γ 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 β subunit in addition to the diol dehydratase-specific extra-regions of β and γ 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 N-terminal sequences are sufficient to change the solubility of the enzyme.

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Abbreviations

AdoCbl:

Adenosylcobalamin or coenzyme B12

PAGE:

Polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

SDS:

Sodium dodecyl sulfate

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Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research ((B) 13480195 and (B) 17370038 and Priority Areas 753 and 513 to T. Toraya and (C) 14580627 to T. Tobimatsu) from the Japan Society for Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Grant of Natural Sciences Research Assistance (to T. Toraya) from the Asahi Glass Foundation, Tokyo, Japan. We would like to thank Dr. Koichi Mori for providing the plasmid pNEX. We thank Ms. Yukiko Kurimoto for her assistance in manuscript preparation.

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Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama, 700-8530, Japan

    Takamasa Tobimatsu, Tsuneo Nishiki, Masaya Morimoto, Ryou Miyata & Tetsuo Toraya

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  1. Takamasa Tobimatsu
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  2. Tsuneo Nishiki
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  3. Masaya Morimoto
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  4. Ryou Miyata
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  5. Tetsuo Toraya
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Correspondence to Tetsuo Toraya.

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Communicated by Theo Hansen.

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Tobimatsu, T., Nishiki, T., Morimoto, M. et al. Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B12-dependent glycerol and diol dehydratases. Arch Microbiol 191, 199–206 (2009). https://doi.org/10.1007/s00203-008-0443-x

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  • DOI: https://doi.org/10.1007/s00203-008-0443-x

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