Basic study for the bioluminescence reaction on insect luciferases

昆虫荧光素酶生物发光反应的基础研究

• 批准号: 12045229
• 负责人: OHMIYA Yoshihiro
• 金额: $ 4.1万
• 依托单位: Shizuoka University The National Institute for Advanced Industrial Science and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas (A)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12045229/
• 关键词: Active site; Bioluminescence; Light color; Luciferase; Luciferin; Mutants; 生物発光; 発光スペクトル; 構造林能相関; pH酸性

The comparison of click beetle and railroadworm luciferases (pH-insensitive) with firefly luciferases (pH-sensitive) showed a set of conserved residues differing between the two groups which could be involved with the bioluminescence spectra pH sensitivity.The substitution C258V in Pyrocoelia miyako (Pml) firefly luciferase resulted ina red-shifted mutant (λmax= 570 nm). In contrast, the substitution V255C in Ragophthalmus ohbai railroad worm luciferase (Rol) had no effect. Substitution of Thr226 in the green light emitting luciferases of Rol and Pyrearinus termitilluminans (Pyt) click beetle luciferases resulted in red-shifts (12 to 35 nm), whereas the substitution T226N in the red light emitting luciferase of Phrixothrix hirtus (PhRE) railroadworm resulted in a 10 nm blue-shift. In PmL the substitution N230S resulted in a typical red mutant (λ= 611nm). The bioluminescence spectrum of all these luciferase mutants did not showed altered pH-sensitivity nor considerably changed half-bandwidth in relation to the wild-type luciferases. Altogether present data suggest that Thr226 is an important residue for keeping active-site core in both groups of beetle luciferases. The mechanism for bioluminescence color determination between pH-sensitive and pH-insensitive luciferases could be different.

点击甲虫和铁路虫荧光素酶（不敏感）与萤火虫荧光素酶（pH敏感）的比较表明，两组之间的一组保守残留物可能与生物发光光谱pH敏感性有关。 nm）。相反，Ragophthalmus Ohbai铁路蠕虫荧光素酶（ROL）中的替代V255C无效。在绿灯中替换绿灯和雷素的荧光素酶（Pyt）单击甲虫荧光素酶会导致红移（12至35 nm），而替代T226N在红光下以红光发光的荧光液（Phrixhixhirix hirtus hirtus hirtus shift yever）blue shift in y nift a railoady nm inm a inm sy a inm a inm a inm sy a inm a inm sy Inf。在PML中，替代N230S导致典型的红色突变体（λ= 611 nm）。所有这些荧光素酶突变体的生物发光光谱都没有显示出改变的pH敏感性，也没有考虑到与野生型荧光素酶相关的半频宽。总共当前的数据表明，THR226是在两组甲虫荧光素酶中保持主动位置的重要居住。 pH敏感和pH不敏感的荧光素酶之间生物发光颜色测定的机制可能不同。

项目成果

Viviani VR, Joaquim Da Silva Neto A, Ohmiya Y: "The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence."Protein Eng Des Sel. 17. 113-117 (2004)

Viviani VR、Joaquim Da Silva Neto A、Ohmiya Y：“残基 220 和 344 之间及以后的区域对 Phrixotrix 铁路蠕虫荧光素酶绿色和红色生物发光的影响。”Protein Eng Des Sel。

VR Viviani: "Bioluminescence color determinants of Phrixothrix railroad-worm luciferases"Photochem.Photobiol. 72. 267-271 (2000)

VR Viviani：“Phrixothrix 铁路蠕虫荧光素酶的生物发光颜色决定因素”Photochem.Photobiol。

Viviani VR., Uchida A., Viviani W, Ohmiya Y: "The influence of Ala243 (Gly247), Arg 215 and Thr 226 (Asn230) on the bioluminescecen spectra and pH-sensitivity of Railroad worm, clicl beetle and firefly luciferases"Photochem.Photobiol. 76. 538-544 (2002)

Viviani VR.、Uchida A.、Viviani W、Ohmiya Y：“Ala243 (Gly247)、Arg 215 和 Thr 226 (Asn230) 对铁路蠕虫、clicl 甲虫和萤火虫荧光素酶的生物发光光谱和 pH 敏感性的影响”Photochem

Viviani VR., Uchida A., Viviani W, Ohmiya Y.: "The influence of Ala243 (Gly247), Arg215 and Thr 226 (Asn230) on the bioluminescecen spectra and pH-sensitivity of Railroad worm, clicl beetle and firefly luciferases."Photochem. Photobiol. 76. 538-544 (2002)

Viviani VR.、Uchida A.、Viviani W、Ohmiya Y.：“Ala243 (Gly247)、Arg215 和 Thr 226 (Asn230) 对铁路蠕虫、clicl 甲虫和萤火虫荧光素酶的生物发光光谱和 pH 敏感性的影响。”

Y.Torahashi: "Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase"And.Biochemi.. 289. 260-266 (2001)

Y.Torahashi：“通过分泌型生物发光报告基因 Vargula hilgendorfii 荧光素酶连续测量靶向启动子活性”And.Biochemi.. 289. 260-266 (2001)