Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z.
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 degrees C with 10 microM 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.
Miyamoto
Emi
E
Department of Health and Nutrition, Nagasaki International University, Sasebo, Japan.
Tanioka
Yuri
Y
Nishizawa-Yokoi
Ayako
A
Yabuta
Yukinori
Y
Ohnishi
Kouhei
K
Misono
Haruo
H
Shigeoka
Shigeru
S
Nakano
Yoshihisa
Y
Watanabe
Fumio
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001051:Apoenzymes / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003038:Cobamides / Q000378:metabolism; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005056:Euglena gracilis / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008765:Methylmalonyl-CoA Mutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011422:Propionates / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D000596:Amino Acids; D001051:Apoenzymes; D003038:Cobamides; D018076:DNA, Complementary; D020035:Holoenzymes; D011422:Propionates; D021122:Protein Subunits; D015800:Protozoan Proteins; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide