Bacterial expression and characterization of molluscan IDO-like myoglobin.
The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic alpha- and beta-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His(74), His(288), and His(332) were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His(332) is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His(74) and His(288) might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.
Yuasa
Hajime Julie
HJ
Laboratory of Biochemistry, Chairs of Bioactive Molecular Technology, Department of Materials Science, Faculty of Science, National University Corporation Kochi University, Kochi 780-8520, Japan. julie@kochi-u.ac.jp
Hasegawa
Tetsuo
T
Nakamura
Takahiro
T
Suzuki
Tomohiko
T
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D049851:Gastropoda / Q000201:enzymology*; D005786:Gene Expression Regulation; D006639:Histidine / Q000235:genetics; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000235:genetics
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D009211:Myoglobin; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D006639:Histidine; D001120:Arginine; D000409:Alanine; D010100:Oxygen