Archives of Microbiology

, Volume 192, Issue 6, pp 437–446 | Cite as

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

  • Emi Miyamoto
  • Yuri Tanioka
  • Ayako Nishizawa-Yokoi
  • Yukinori Yabuta
  • Kouhei Ohnishi
  • Haruo Misono
  • Shigeru Shigeoka
  • Yoshihisa Nakano
  • Fumio WatanabeEmail author
Original Paper


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.


Cobalamin Euglena gracilisMethylmalonyl-CoA mutase Photoassimilation Propionate metabolism 











Methylmalonyl-CoA mutase


Polyvinylidene difluoride


Sodium dodecyl sulfate


  1. Ackrell BC, Kearney EB, Singer TP (1978) Mammalian succinate dehydrogenase. Meth Enzymol 53:466–483CrossRefPubMedGoogle Scholar
  2. Amero SA, James TC, Elgin SCR (1988) Production of antibodies using proteins in gel bands. In: Walker JM (ed) Methods in molecular biology, vol 3. Humana Press, New Jersey, pp 355–362Google Scholar
  3. Bairoch A, Bucher P, Hofman K (1997) The PROSITE database, its status in 1997. Nucleic Acids Res 25:217–221CrossRefPubMedGoogle Scholar
  4. Charles TC, Aneja P (1999) Methylmalonyl-CoA mutase encoding gene of Sinorhizobium meliloti. Gene 226:121–127CrossRefPubMedGoogle Scholar
  5. Cramer MC, Myers J (1952) Growth and photosynthetic characterisitics of Euglena gracilis. Arch Microbiol 17:384–402Google Scholar
  6. Dobson CM, Wai T, Leclerc D, Wilson A, Wu X, Dore C, Hudson T, Rosenblatt DS, Gravel RA (2002) Identification of the gene responsible for the cblA complementation group of vitamin B12-responsive methylmalonic acidemia based on analysis of prokaryotic gene arrangements. Proc Natl Acad Sci USA 99:15554–15559CrossRefPubMedGoogle Scholar
  7. Drennan CL, Huang S, Drummond JT, Matthews RG, Ludwig ML (1994a) How a protein binds B12: A 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266:1669–1674CrossRefPubMedGoogle Scholar
  8. Drennan CL, Matthews RG, Ludwig ML (1994b) Cobalamin-dependent methionine synthase: the structure of a methylcobalamin-binding fragment and implications for other B12-dependent enzymes. Curr Opin Struct Biol 4:919–929CrossRefPubMedGoogle Scholar
  9. Fenton WA, Hack AM, Willard HF, Gertler A, Rosenberg LE (1982) Purification and properties of methylmalonyl coenzyme a mutase from human liver. Arch Biochem Biophys 214:815–826CrossRefPubMedGoogle Scholar
  10. Francalanci F, Davis NK, Fuller JQ, Murfitt D, Leadlay PF (1986) The subunit structure of methylmalonyl-CoA mutase from Propionibacterium shermanii. Biochem J 236:489–494PubMedGoogle Scholar
  11. Frantz C, Ebel C, Paulus F, Imbaut P (2000) Characterization of trans-splicing in Euglenoids. Curr Genet 37:349–355CrossRefPubMedGoogle Scholar
  12. Gaire D, Sponne I, Droesch S, Charlier A, Nicolas JP, Lambert D (1999) Comparison of two methods for the measurement of rat liver methylmalonyl-coenzyme a mutase activity: HPLC and radioisotopic assays. J Nutr Biochem 10:56–62CrossRefPubMedGoogle Scholar
  13. Han YS, Bratt JM, Hogenkamp HPC (1984) Purification and characterization of methylmalonyl-CoA mutase from Ascaris lumbricodes. Comp Biochem Physiol 78B:41–45Google Scholar
  14. Isegawa Y, Nakano Y, Kitaoka S (1984) Conversion and distribution of cobalamin in Euglena gracilis Z, with special reference to its location and probable function within chloroplasts. Plant Physiol 76:814–818CrossRefPubMedGoogle Scholar
  15. Ishikawa T, Tajima N, Nishikawa H, Gao Y, Rapolu M, Shibata H, Sawa Y, Shigeoka S (2010) Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric structure: its unique molecular characterization. Biochem J 426:125–134CrossRefPubMedGoogle Scholar
  16. Jansen R, Kalousek F, Fenton WA, Rosenberg LE, Ledley FD (1989) Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction. Genomics 4:198–205CrossRefPubMedGoogle Scholar
  17. Kellermeyer RW, Allen SHG, Stjernhorm R, Wood HG (1964) Methylmalonyl Isomerase. J Biol Chem 239:2562–2569PubMedGoogle Scholar
  18. Kitaoka S, Nakano Y, Miyatake K, Yokota A (1989) Enzymes and their functional location. In: Buetow DE (ed) The biology of euglena, vol 4. Academic Press, San Diego, pp 1–135Google Scholar
  19. Korotkova N, Lidstrom ME (2004) MeaB is a component of the methylmalonyl-CoA mutase complex required for protection of the enzyme from inactivation. J Biol Chem 279:13652–13658CrossRefPubMedGoogle Scholar
  20. Ludwig ML, Drennan CL, Matthews RG (1996) The reactivity of B12 cofactors: the proteins make a difference. Structure 4:505–512CrossRefPubMedGoogle Scholar
  21. Marsh ENG, Holloway DE (1992) Cloning and sequencing of glutamate mutase component S from Clostridium tetanomorphum Homologies with other cobalamin-dependent enzymes. FEBS Lett 310:167–170CrossRefPubMedGoogle Scholar
  22. Miyamoto E, Watanabe F, Yamaji R, Inui I, Sato K, Nakano Y (2002) Purification and characterization of methylmalonyl-CoA mutase from a ethanol-utilizing bacterium, Methylobacterium extorquens NR-1. J Nutr Sci Vitaminol 48:242–246PubMedGoogle Scholar
  23. Miyamoto E, Watanabe F, Charles TC, Yamaji R, Inui H, Nakano Y (2003) Purification and characterization of homodimeric methylmalonyl-CoA mutase from Sinorhizobium meliloti. Arch Mirobiol 180:151–154CrossRefGoogle Scholar
  24. Miyamoto E, Watanabe F, Yamaguchi Y, Takenaka H, Nakano Y (2004) Purification and characterization of methylmalonyl-CoA mutase from a photosynthetic coccolithophorid alga, Pleurochrysis carterae. Comp Biochem Physiol B 138:163–167CrossRefPubMedGoogle Scholar
  25. Miyamoto E, Tanioka Y, Yukino Y, Hayashi M, Watanabe F, Nakano Y (2007) Occurrence of 5′-deoxyadenosylcobalamin and its physiological function as the coenzyme of metnylmalonyl-CoA mutase in a marine eukaryotic microorganism, Schizochytrium limacinum SR21. J Nutr Sci Vitaminol 53:471–475CrossRefPubMedGoogle Scholar
  26. Padovani D, Banerjee R (2006) Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone. Biochemistry 45:9300–9306CrossRefPubMedGoogle Scholar
  27. Tessier LH, Keller M, Chan RL, Fournier R, Weil JH, Imbault P (1991) Short leader sequences may be transferred from small RNAs to pre-mature mRNA by trans-splicing in Euglena. EMBO J 10:2621–2625PubMedGoogle Scholar
  28. Tokunaga M, Nakano T, Kitaoka S (1976) Separation and properties of the NAD-linked and NADP-linked isozymes of succinic semialdehyde dehydrogenase in Euglena gracilis Z. Biochim Biophys Acta 429:55–62PubMedGoogle Scholar
  29. Watanabe F, Nakano Y, Kitaoka S (1987) Isolation and some properties of soluble and membrane-bound cobalamin binding proteins of Euglena mitochondira. Arch Microbiol 149:30–35CrossRefGoogle Scholar
  30. Watanabe F, Nakano Y, Stupperich E (1992) Different corrinoid specificities for cell growth and the cobalamin uptake system in Euglena gracilis Z. J Gen Microbiol 138:1807–1813Google Scholar
  31. Watanabe F, Tamura Y, Stupperich E, Nakano Y (1993) Uptake of cobalamin by Euglena mitochondria. J Biochem 114:793–799PubMedGoogle Scholar
  32. Watanabe F, Abe K, Tamura Y, Nakano Y (1996) Adenosylcobalamin-dependent methylmalonyl-CoA mutase isozymes in the photosynthetic protozoon Euglena gracilis Z. Microbiology 142:2631–2634CrossRefPubMedGoogle Scholar
  33. Wilkemeyer MF, Crane AM, Ledley FD (1990) Primary structure and activity of mouse methylmalonyl-CoA mutase. Biochem J 271:449–455PubMedGoogle Scholar
  34. Yokota A, Hosotani K, Kitaoka S (1982) Mechanism of metabolic regulation in photoassimilation of propionate in Euglena gracilis Z. Arch Biochem Biophys 249:530–537CrossRefGoogle Scholar
  35. Zeng S, Gong Z (2002) Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary. Gene 294:45–53CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Emi Miyamoto
    • 2
  • Yuri Tanioka
    • 3
    • 7
  • Ayako Nishizawa-Yokoi
    • 4
  • Yukinori Yabuta
    • 1
  • Kouhei Ohnishi
    • 5
  • Haruo Misono
    • 6
  • Shigeru Shigeoka
    • 4
  • Yoshihisa Nakano
    • 3
  • Fumio Watanabe
    • 1
    Email author
  1. 1.Faculty of Agriculture, School of Agricultural, Biological and Environmental SciencesTottori UniversityTottoriJapan
  2. 2.Department of Health and NutritionNagasaki International UniversitySaseboJapan
  3. 3.Graduate School of Life and Environmental SciencesOsaka Prefecture UniversitySakaiJapan
  4. 4.Faculty of Agriculture, Department of Advanced BioscienceKinki UniversityNaraJapan
  5. 5.Research Institute of Molecular GeneticsKochi UniversityKochiJapan
  6. 6.Faculty of AgricultureKochi UniversityKochiJapan
  7. 7.Department of NutritionJunior College of Tokyo University of AgricultureTokyoJapan

Personalised recommendations