Structure of P. gingivalis SOD analyzed by mutagenesis and molecular evolution

通过诱变和分子进化分析牙龈卟啉单胞菌 SOD 的结构

• 批准号: 12671819
• 负责人: HIRAOKA B.yukihiro
• 金额: $ 1.92万
• 依托单位: Matsumoto Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671819/
• 关键词: Reactive oxygen; SOD; Superoxyde dismutase; Metallo enzyme; Oxidoreductase; Porphyromanas gingivalis; Metal-specific activity; Site-directed mutagenesis; Porphyromonas gingivalis

Active site of Mn and Fe-superoxide dismutases (SODs) have very similar primary and tertiary structure, however, the expression of enzymatic activity of most SODs have strict specificity for the metal at the active site. While P. gingivalies(P.g.) SOD shows exceptionally activity with either Mn or Fe incorporated into the same active site. We have prepared the mutant of the P.g.-SOD with conversions of above amino acid residues to Mn- or Fe-type, in order to clarify contribution of these residues to the metal-specific acitivty of P.g.-SOD.[RESULTS] We made the Mn- and Fe-type mutation, by site-directed mutagenesis and expressed as a soluble fusion protein coupled to maltose binding protein (MBP) using the expression vector pMAL-c2. After an affinity purification and cleavage MBP by trypsin, the enzyme was purified by anion exchange chromatography with yield of 15-20 mg/l of culture. Wild, the Mn- and Fe-type mutant enzymes were reconstituted with Mn or Fe by using an acid-guanidine-HCl method and purified to homogeneous by a hyroxyapatite-HPLC. The ratio of the specific activity of Mn-/Fe-reconstituted SOD was about 1.5 in the native SOD, to 2.4 in the Mn-type mutant SOD and to 13.4 in the Fe-type mutant SOD. In next study, molecular evolution maybe useful to search for unknown amino acid residues which contribute to the metal-specific activity of Mn-, Fe- and cambialistic SODs.

Mn和氧化二氧化碳歧化酶（SOD）的活性位点具有非常相似的主要和三级结构，但是，大多数SOD的酶促活性的表达对活性位点的金属具有严格的特异性。而P.牙龈（P.G。）SOD显示出与MN或Fe掺入同一活性位点的非凡活性。我们已经用上述氨基酸残基向Mn-或Fe-type的转化准备了P.G.-SOD的突变体，以阐明这些残基对P.G.-SOD的金属特异性依赖的贡献。使用表达矢量PMAL-C2。在用胰蛋白酶进行亲和力纯化和切割MBP之后，该酶通过阴离子交换色谱法纯化，产量为15-20 mg/l培养。使用酸 - 瓜氨酸-HCL方法将野生，Mn-和Fe-Type突变酶与MN或FE重构，并通过氢氧基磷灰石-HPLC纯化为均匀。在天然SOD中，Mn-/Fe重新确定的SOD的特异性活性的比率约为1.5，在MN型突变体SOD中为2.4，而在Fe-Type突变SOD中的比率约为13.4。在下一项研究中，分子进化可能有助于寻找未知的氨基酸残基，这些氨基酸残基有助于Mn-，Fe-和cambialistic SOD的金属特异性活性。

项目成果

Hiraoka, B.Y., Sugio, S., Kobayashi, K. and Yamakura, F.: "Change of metal-specific activity of Fe/Mn-superoxide dismutase by mutation of amino acid residues. (Japanese)"The 1st anuual meeting of the protein science society of japan : Program and abstract

Hiraoka, B.Y.、Sugio, S.、Kobayashi, K. 和 Yamakura, F.：“通过氨基酸残基突变改变铁/锰超氧化物歧化酶的金属比活性。（日语）”第一次年会

Hiraoka, B.Y., Yamakura, F., Sugio, S., Nakayama, K.: "A change of the metal-specific activity of a cambialistic superoxide dismutase from Porphyromonas gingivalis by a double mutation of Gln-70 to Gly and Ala-142 to Gin"Biochem.J.. 345・2. 345-350 (2000)

Hiraoka, B.Y.、Yamakura, F.、Sugio, S.、Nakayama, K.：“通过 Gln-70 到 Gly 和 Ala-142 的双重突变，改变牙龈卟啉单胞菌的形成性超氧化物歧化酶的金属特异性活性至杜松子酒“Biochem.J.. 345・2. 345-350 (2000)

平岡行博: "P.gingivalisSODの金属寛容性にGly155が関与している"歯基礎誌. 43・5. 605 (2001)

Yukihiro Hiraoka：“Gly155 与牙龈卟啉单胞菌 SOD 的金属耐受性有关”《牙科科学杂志》43・5（2001 年）。

Hiraoka, B.Y., Yamakura, R, Sugio, S. and Nakayama, K.: "A chenge of the metal-specific activity of a cambialistic superoxide dismutase from Porohyromonas gingivalis by a double mutation of Gln-70 to Gly and Ala-142 to Gin."Biochemical J.. 345-2. 345-350

Hiraoka, B.Y.、Yamakura, R、Sugio, S. 和 Nakayama, K.：“通过 Gln-70 到 Gly 和 Ala-142 的双重突变，对牙龈多孔单胞菌的形成性超氧化物歧化酶的金属特异性活性进行了改造。

平岡行博, 杉尾成俊, 小林一雄, 山倉文幸: "アミノ酸変異によるFe/Mn-スーパーオキシドジスムターゼの活性金属特異性の変換"第1回日本蛋白質科学会年会 プログラム・要旨集. 313 (2000)

Yukihiro Hiraoka、Shigetoshi Sugio、Kazuo Kobayashi、Fumiyuki Yamakura：“通过氨基酸突变转换铁/锰超氧化物歧化酶的活性金属特异性”日本蛋白质科学学会第一届年会计划和摘要 313 (2000)。