Cloning and characterization of Chlorobium tepidum ferritin.
The Chlorobium tepidum ferritin (CtFtn) gene was synthesized and cloned into a pET3a expression vector (Novagen). CtFtn was expressed in Escherichia coli and purified to electrophoretic homogeneity. Sequence analysis indicates that all the conserved amino acids required to form the Fe(2+) oxidizing ferroxidase center are present. Ftn is highly conserved from bacteria to humans, each subunit folds into a 4-helical bundle (helices A-D), with a long loop connecting helices B and C, plus a fifth short E-helix at the C-terminus. Calculations based on the secondary structure of CtFtn predict that each of these helices forms. However, the sequence of CtFtn shows a much longer C-terminus with a significant number of polar amino acids. Size-exclusion chromatography shows that CtFtn elutes at a size consistent with a 24-subunit protein cage. Incubation of CtFtn with Fe(2+) produced an increase in the absorbance at 310 nm consistent with the incorporation of iron inside CtFtn. Assays monitoring ferroxidase activity showed that CtFtn possesses ferroxidase activity but it is less active than human H-chain ferritin. Additionally, the iron loading capacity of CtFtn is significantly reduced compared to proteins from other organisms. We propose that the unique extended C-terminus in CtFtn causes the decreased iron loading in CtFtn and possibly influences the slower rate of iron oxidation at the ferroxidase center.
Instituto de Agroindustria, Facultad de Ciencias Agropecuarias y Forestales, Universidad de la Frontera, Temuco, Chile.
Alejandro E
AE
Yevenes
Valeria
V
Marquez
Richard K
RK
Watt
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D041883:Chlorobium; D003001:Cloning, Molecular; D005293:Ferritins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment
D001426:Bacterial Proteins; D005293:Ferritins; D007501:Iron; D010100:Oxygen