Mutational analysis of the pea phytochrome A chromophore pocket: chromophore assembly with apophytochrome A and photoreversibility.
Mutational analysis of the pea phytochrome A chromophore pocket: chromophore assembly with apophytochrome A and photoreversibility.
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豌豆光敏色素 A 生色团口袋的突变分析：与光敏色素 A 和光可逆性的生色团组装。
DOI：
10.1021/bi00214a014
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Song,PS
中科院分区：
文献类型：
--
作者：
Deforce,L;Furuya,M;Song,PS
修订稿于 1993 年 10 月 26 日收到®摘要：在酿酒酵母中表达豌豆脱辅基色素 A 的十个位点特异性突变体，并分析脱辅基蛋白的发色团组装和光可逆吸光度变化。突变体对位于发色团附着残基（豌豆光敏色素 A 中的 Cys-323）微环境中的五个保守氨基酸残基中的每一个构成两个特定变化。所有突变体脱辅基光色素均能够自催化共价附着藻蓝蛋白，表明脱辅基蛋白中不存在主要的结构扰动。然而，突变体之间的发色团连接率差异显着。从光谱上看，突变型全光敏色素分为三种类型：与野生型加合物无法区分的突变型光敏色素、与野生型加合物相比具有蓝移 Pr 和 Pfr 吸收最大值的突变体以及不可光逆的突变体。通过对结果的分析，我们得出结论，残基 Asp-309、Arg-318、His-321 和 Gln-326 可能不催化参与发色团连接反应，但某些残基可能发挥重要的结构和立体化学作用。正如所建议的，Arg-318 可能锚定发色团 [Partis, M. D., & Grimm, R.(1990) Z. Naturforsch. 45c，987-998； Parker, W., et al.(1993) Bioconjugate Chem.（印刷中）]。保守的 Gln-326 是发色团附着位点下游的三个残基，对于光敏色素的光谱完整性和光可逆性而言，在静电方面并不重要，但该残基对于裂解酶活性在空间上很重要。看来，发色团结合 Cys-323 的 N 端和 C 端附近的 5 个氨基酸残基对连接反应的作用是结构性的，而不是催化性的。植物光形态发生的许多方面是由光敏色素（红色/远红光可逆光受体家族）控制的（Thomas & Johnson，1991；Furuya，1993）。豌豆光敏色素 A (PhyA1) 是两个相同亚基的同二聚体，分子量为 *= 121 kDa（Nakasako 等人，1990）。每个亚基都带有一个共价结合的四吡咯发色团（Lagarias & Rapoport，1980）。胆素发色团通过硫醚键与位于脱辅基蛋白 N 末端一半的单个半胱氨酸硫醇以立体特异性方式连接到脱辅基蛋白 (Lagarias & Rapoport, 1980)。 PhyA 在黑暗生长的豌豆幼苗中以其吸收红光的形式（Pr.）积累。发色团吸收光后，发色团的 C 15 -C 16 双键周围发生顺式/反式异构化（Rudiger 等人，1983；Rospendowski 等人，1989；Fodor 等人，1990）。这会引起脱辅基蛋白和发色团的构象变化[Sommer 和 Song (1990) 及其参考文献]。这个的最终产品
Revised Manuscript Received October 26, 1993® abstract: Ten site-specific mutants of pea apophytochrome A were expressed in Saccharomycescerevisiae and analyzed for chromophore assembly with apoprotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of Five conserved amino acid residues located in the microenvironment of the chromophore attachment residue, which is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicating that there were no major structural perturbations in the apoproteins. However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three types: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted Pr and Pfr absorption maxima compared to the wild-type adduct, and mutants that are not photoreversible. From an analysis of the results, we concluded that the residues Asp-309, Arg-318, His-321, and Gln-326 are probably not catalytically involved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M. D., & Grimm, R.(1990) Z. Naturforsch. 45c, 987-998; Parker, W., et al.(1993) Bioconjugate Chem.(in press)]. Theconserved Gln-326, threeresidues downstream from the chromophore attachment site, is not electrostatically critical for the spectral integrity and photoreversibility of phytochrome, but this residue is sterically important to thelyase activity. It appears that the role of the five amino acid residues in the N-and C-terminal vicinities of the chromophore binding Cys-323 is structural rather than catalytic for the ligation reaction.Many aspects of plant photomorphogenesis are controlled by the phytochromes, a family of red/far-red photoreversible light receptors (Thomas & Johnson, 1991; Furuya, 1993). Pea phytochrome A (PhyA1) is a homodimer of two identical subunits with a molecular mass of*= 121 kDa (Nakasako et al., 1990). Each subunit bears a covalently bound tetrapyrrolic chromophore (Lagarias & Rapoport, 1980). The bilin chromophore is ligated to the apoprotein in a stereospecific fashion through a thioether bond with a single cysteine thiol, located in the N-terminal half of the apoprotein (Lagarias & Rapoport, 1980). PhyA accumulates in dark-grown pea seedlings in its red light absorbing form, Pr. Following absorption of light by the chromophore, a cis/trans isomerization around the Cj5-Ci6 double bond of the chromophore takes place (Rudiger et al., 1983; Rospendowski et al., 1989; Fodor et al., 1990). This induces conformational changes in both the apoprotein and the chromophore [Sommer and Song (1990) and references therein]. The final product of this