Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr.
Glycine 165, which is located near the active site metal, is mostly conserved in aligned amino acid sequences of manganese-containing superoxide dismutase (Mn-SOD) proteins, but is substituted to threonine in most iron-containing SODs (Fe-SODs). Because threonine 165 is located between Trp128 and Trp130, and Trp128 is one of the metal-surrounding aromatic amino acids, the conversion of this amino acid may affect the metal-specific activity of Escherichia coli Mn-SOD. In order to clarify this possibility, we prepared a mutant of E. coli Mn-SOD with the replacement of Gly165 by Thr. The ratio of the specific activities of Mn- to Fe-reconstituted enzyme increased from 0.006 in the wild-type to 0.044 in the mutant SOD; therefore, the metal-specific SOD was converted to a metal-tolerant SOD. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs indicated the loss of Mn-SOD character. It was concluded that Gly at position 165 plays a catalytic role in maintaining the integrity of the metal specificity of Mn-SOD.
Osawa
Masaki
M
Department of Hard Tissue Research, Graduate School of Oral Medicine, Matsumoto Dental University, Shiojiri 3990781, Japan.
Yamakura
Fumiyuki
F
Mihara
Masashi
M
Okubo
Yuichiro
Y
Yamada
Kazuhiro
K
Hiraoka
B Yukihiro
BY
D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005998:Glycine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008345:Manganese / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D013912:Threonine; D008345:Manganese; D013482:Superoxide Dismutase; D005998:Glycine