Pronounced conversion of the metal-specific activity of superoxide dismutase from Porphyromonas gingivalis by the mutation of a single amino acid (Gly155Thr) located apart from the active site.
Glycine 155, which is located approximately 10 A from the active metal sites, is mostly conserved in aligned amino acid sequences of manganese-specific superoxide dismutases (Mn-SODs) and cambialistic SOD (showing the same activity with Fe and Mn) from Porphyromonas gingivalis, but is substituted for threonine in most Fe-SODs. Since Thr155 is located between Trp123 and Trp125, and Trp123 is one member of the metal-surrounding aromatic amino acids, there is a possibility that the conversion of this amino acid may cause a conversion of the metal-specific activity of cambialistic P. gingivalis SOD. To clarify this possibility, we have prepared a mutant of the P. gingivalis SOD with conversion of Gly155 to Thr. The ratios of the specific activities of Fe- to Mn-reconstituted enzyme, which are measured by the xanthine oxidase/cytochrome c method, increased from 0.6 in the wild-type to 11.2 in the mutant SODs, indicating the conversion of the metal-specific activity of the enzyme from a cambialistic type to an Fe-specific type. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs closely resembled those of Fe-specific SOD. Furthermore, the EPR spectra of the Fe- and Mn-reconstituted mutant SODs also closely resembled those of Fe-specific SOD. Three-dimensional structures of the Fe-reconstituted wild-type SOD and Mn-reconstituted mutant SOD have been determined at 1.6 A resolution. Both structures have identical conformations, orientations of residues involved in metal binding, and hydrogen bond networks, while the side chain of Trp123 is moved further toward the metal-binding site than in wild-type SOD. A possible contribution of the structural differences to the conversion of the metal-specific activity through rearrangement of the hydrogen bond network among Trp123, Gln70, Tyr35, and the metal-coordinated solvent is discussed.
Yamakura
Fumiyuki
F
Department of Chemistry, Juntendo University School of Medicine, Inba 270-1695, Japan. yamakura@sakura.juntendo.ac.jp
Sugio
Shigetoshi
S
Hiraoka
B Yukihiro
BY
Ohmori
Daijiro
D
Yokota
Takehiro
T
D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013912:Threonine; D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D005998:Glycine