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Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z

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Abstract

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with 10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS–polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

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Abbreviations

AdoCbl:

5′-deoxyadenosylcobalamin

AdoCbi:

5′-deoxyadenosylcobinamide

Cbl:

Cobalamin

CN-Cbl:

Cyanocobalamin

MCM:

Methylmalonyl-CoA mutase

PVDF:

Polyvinylidene difluoride

SDS:

Sodium dodecyl sulfate

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Author information

Author notes

  1. Yuri Tanioka

    Present address: Department of Nutrition, Junior College of Tokyo University of Agriculture, Tokyo, Japan

Authors and Affiliations

  1. Faculty of Agriculture, School of Agricultural, Biological and Environmental Sciences, Tottori University, Tottori, Japan

    Yukinori Yabuta & Fumio Watanabe

  2. Department of Health and Nutrition, Nagasaki International University, Sasebo, Japan

    Emi Miyamoto

  3. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan

    Yuri Tanioka & Yoshihisa Nakano

  4. Faculty of Agriculture, Department of Advanced Bioscience, Kinki University, Nara, Japan

    Ayako Nishizawa-Yokoi & Shigeru Shigeoka

  5. Research Institute of Molecular Genetics, Kochi University, Kochi, Japan

    Kouhei Ohnishi

  6. Faculty of Agriculture, Kochi University, Kochi, Japan

    Haruo Misono

Authors
  1. Emi Miyamoto
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  2. Yuri Tanioka
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  3. Ayako Nishizawa-Yokoi
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  4. Yukinori Yabuta
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  5. Kouhei Ohnishi
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  6. Haruo Misono
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  7. Shigeru Shigeoka
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  8. Yoshihisa Nakano
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  9. Fumio Watanabe
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Corresponding author

Correspondence to Fumio Watanabe.

Additional information

Communicated by Erko Stackebrandt.

The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number AB443637.

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Miyamoto, E., Tanioka, Y., Nishizawa-Yokoi, A. et al. Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z. Arch Microbiol 192, 437–446 (2010). https://doi.org/10.1007/s00203-010-0572-x

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  • DOI: https://doi.org/10.1007/s00203-010-0572-x

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