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

• Published in: Biochemistry (Easton) 2003-09, Vol.42 (36), p.10790-10799
• Main Authors: Yamakura, Fumiyuki, Sugio, Shigetoshi, Hiraoka, B. Yukihiro, Ohmori, Daijiro, Yokota, Takehiro
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Binding Sites; Crystallography, X-Ray; Electron Spin Resonance Spectroscopy; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Glycine - genetics; Glycine - metabolism; Humans; Hydrogen Bonding; Iron - chemistry; Iron - metabolism; Manganese - chemistry; Manganese - metabolism; Models, Molecular; Porphyromonas gingivalis; Porphyromonas gingivalis - enzymology; Protein Conformation; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spectrophotometry - methods; Substrate Specificity; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; Threonine - genetics; Threonine - metabolism; Xanthine Oxidase - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0349625

Glycine 155, which is located ∼10 Å 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 Å 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.