Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum.
Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum.
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DOI：
10.3390/ijms231911716
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发表时间：
2022-10-03
影响因子：
5.6
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中科院分区：
文献类型：
--
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叶绿素酶（chlorophyllase，CLH）是一种早被发现的叶绿素脱植基化酶，在叶片衰老过程中不起作用。然而，CLH被认为参与水果的降解，并对外部和激素刺激作出反应。本研究的目的是详细阐明番茄基因组中4种叶绿素酶的生化、结构特性和基因表达，以了解番茄叶绿素酶（Solanum lycopersicum chlorophyllases，SLCLH）的作用。SlCLH 1/4基因在叶片和果实发育成熟期表达量最高，SlCLH 1基因在成熟绿色果实中受光调控。SlCLH 1/2/3/4含有高度保守的GHSXG脂肪酶基序和Ser-Asp-His催化三联体。我们确定Ser 159，Asp 226，和His 258作为必要的催化三联体的定点诱变重组SlCLH 1。动力学分析表明，SlCLH 1对叶绿素a、叶绿素B和脱镁叶绿素a（Phein a）具有较高的水解活性，但对叶绿素a和叶绿素B的水解活性高于对Phein a的水解活性; SlCLH 2/3对叶绿素a和叶绿素B的水解活性很低，而SlCLH 4不具有叶绿素脱镁叶绿素活性。重组SlCLH 1/2/3具有不同的pH稳定性和最适温度。去除预测的N-末端加工肽导致重组SlCLH 1/2活性的部分损失，但不损害SlCLH 3活性。这些不同的特性暗示它们在番茄中可能具有不同的生理功能。
Recent studies have confirmed that chlorophyllase (CLH), a long-found chlorophyll (Chl) dephytylation enzyme for initiating Chl catabolism, has no function in leaf senescence-related Chl breakdown. Yet, CLH is considered to be involved in fruit degreening and responds to external and hormonal stimuli. The purpose of this work was to elucidate in detail the biochemical, structural properties, and gene expression of four CLHs from the Solanum lycopersicum genome so as to understand the roles of Solanum lycopersicum chlorophyllases (SlCLHs). SlCLH1/4 were the predominantly expressed CLH genes during leaf and fruit development/ripening stages, and SlCLH1 in mature green fruit was modulated by light. SlCLH1/2/3/4 contained a highly conserved GHSXG lipase motif and a Ser-Asp-His catalytic triad. We identified Ser159, Asp226, and His258 as the essential catalytic triad by site-directed mutagenesis in recombinant SlCLH1. Kinetic analysis of the recombinant enzymes revealed that SlCLH1 had high hydrolysis activities against Chl a, Chl b, and pheophytin a (Phein a), but preferred Chl a and Chl b over Phein a; SlCLH2/3 only showed very low activity to Chl a and Chl b, while SlCLH4 showed no Chl dephytylation activity. The recombinant SlCLH1/2/3 had different pH stability and temperature optimum. Removal of the predicted N-terminal processing peptide caused a partial loss of activity in recombinant SlCLH1/2 but did not compromise SlCLH3 activity. These different characteristics among SlCLHs imply that they may have different physiological functions in tomato.
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