Glutathione S-transferases （GSTs） are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana （AtGSTZ） can efficiently degrade dichloroacetic acid （DCA）, which is a common carcin...Glutathione S-transferases （GSTs） are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana （AtGSTZ） can efficiently degrade dichloroacetic acid （DCA）, which is a common carcinogenic contaminant in drinking water. Ser73 in AtGSTZ is a conserved residue at Glutathione binding site （G-site）. Compared with the equivalent residues in other GSTs, the catalytic and structural properties of Ser73 were poorly investigated. In this article, site-saturation mutagenesis was performed to characterize the detailed role of Ser73. The DCA dechlorinating （DCA-DC） activity showed that most of the mutants had less than 3% of the wild-type activity, except S73T and S73A showing 43.48% and 21.62% of the wild-type activity, respectively, indicating that position 73 in AtGSTZ showed low mutational substitutability. Kinetic experiments revealed that mutants S73T, S73A, and S73G showed low binding affinity and catalytic efficiency toward DCA, 1.8-, 3.1-, and 10.7- fold increases in KmDcA values and 4.0-, 9.6-, and 34.1- fold decreases in Kcat- DCA/KmDCA values, respectively, compared to the wild type. Thermostability and refolding experiments showed that the wild type mainmined more thermostability and recovered activity. These results demonstrated the important role of Ser73 in catalytic activity and structural stability of the enzyme. Such properties of Ser73 could be particularly crucial to the molecular evolution of AtGSTZ and might, therefore, help explain why Ser73 is conserved in all GSTs. This conclusion might provide insights into the directed evolution of the DCA-DC activity of AtGSTZ.展开更多
The researches have been carried out in 2011-2012, in the experimental fields of Yuryev Plant Production Institute Kharkiv, Ukraine. The forms of ＂00＂ type （a low content of erucic acid and glucosinolates） and sel...The researches have been carried out in 2011-2012, in the experimental fields of Yuryev Plant Production Institute Kharkiv, Ukraine. The forms of ＂00＂ type （a low content of erucic acid and glucosinolates） and self-pollinated lines--110M-10, 127M-10, 132M-10 were studied; the forms of＂0＋＂ type--selfed lines 124-10, 221-10, 305-10; the forms of＂＋0＂ type--cultivars Victor, Janus, Emerald; forms of ＂＋＋＂ type--cultivars Fyedorovskiy, Marens, Uspikh, as well as, self-incompatible forms 5C, 15C-10, 32C-10 and the original sterile form of mutant origin were used. Viability in stigmas of the pistil lasted 3-7 days, in the forms of ＂＋0＂, ＂＋＋＂ type, 7-15 days in the cultivars and forms of ＂0＋＂, ＂00＂ type, 14-21 days in the self-incompatible and sterile forms. A fast loss of sensitivity to pollen by pistil＇s stigma was observed in the forms of＂＋0＂ and ＂＋＋＂ types. On the contrary, in self-incompatible and sterile forms was observed a gradual increase of sensitivity of pistil＇s stigma to pollination and then its gradual fall. A maximum of sensitivity of a pistil＇s stigma to pollen in ＂00＂ and ＂0＋＂ forms was on the 3rd-6th day after castration.展开更多
基金北京市社会科学基金项目“语义地图及语言接触视角下的汉语时体态研究”(编号:16YYC026)香港研究资助局优配研究计划“The Postverbal Constraint across Chinese Dialects”(编号:641913)+1 种基金国家社会科学基金重大项目“功能-类型学取向的汉语语义演变研究”(编号:14ZDB098)北京大学中文系自主科研项目“北方方言的时体范畴研究”(编号:2015ZZKY04)的资助
基金国家自然科学基金(the National Natural Science Foundation of China under Grant No.59735120)国家高技术研究发展计划(863)(theNational High-Tech Research and Development Plan of China under Grant No.200341111)。
基金the National Natural Science Foundation of China (No. 30671183)
文摘Glutathione S-transferases （GSTs） are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana （AtGSTZ） can efficiently degrade dichloroacetic acid （DCA）, which is a common carcinogenic contaminant in drinking water. Ser73 in AtGSTZ is a conserved residue at Glutathione binding site （G-site）. Compared with the equivalent residues in other GSTs, the catalytic and structural properties of Ser73 were poorly investigated. In this article, site-saturation mutagenesis was performed to characterize the detailed role of Ser73. The DCA dechlorinating （DCA-DC） activity showed that most of the mutants had less than 3% of the wild-type activity, except S73T and S73A showing 43.48% and 21.62% of the wild-type activity, respectively, indicating that position 73 in AtGSTZ showed low mutational substitutability. Kinetic experiments revealed that mutants S73T, S73A, and S73G showed low binding affinity and catalytic efficiency toward DCA, 1.8-, 3.1-, and 10.7- fold increases in KmDcA values and 4.0-, 9.6-, and 34.1- fold decreases in Kcat- DCA/KmDCA values, respectively, compared to the wild type. Thermostability and refolding experiments showed that the wild type mainmined more thermostability and recovered activity. These results demonstrated the important role of Ser73 in catalytic activity and structural stability of the enzyme. Such properties of Ser73 could be particularly crucial to the molecular evolution of AtGSTZ and might, therefore, help explain why Ser73 is conserved in all GSTs. This conclusion might provide insights into the directed evolution of the DCA-DC activity of AtGSTZ.
文摘The researches have been carried out in 2011-2012, in the experimental fields of Yuryev Plant Production Institute Kharkiv, Ukraine. The forms of ＂00＂ type （a low content of erucic acid and glucosinolates） and self-pollinated lines--110M-10, 127M-10, 132M-10 were studied; the forms of＂0＋＂ type--selfed lines 124-10, 221-10, 305-10; the forms of＂＋0＂ type--cultivars Victor, Janus, Emerald; forms of ＂＋＋＂ type--cultivars Fyedorovskiy, Marens, Uspikh, as well as, self-incompatible forms 5C, 15C-10, 32C-10 and the original sterile form of mutant origin were used. Viability in stigmas of the pistil lasted 3-7 days, in the forms of ＂＋0＂, ＂＋＋＂ type, 7-15 days in the cultivars and forms of ＂0＋＂, ＂00＂ type, 14-21 days in the self-incompatible and sterile forms. A fast loss of sensitivity to pollen by pistil＇s stigma was observed in the forms of＂＋0＂ and ＂＋＋＂ types. On the contrary, in self-incompatible and sterile forms was observed a gradual increase of sensitivity of pistil＇s stigma to pollination and then its gradual fall. A maximum of sensitivity of a pistil＇s stigma to pollen in ＂00＂ and ＂0＋＂ forms was on the 3rd-6th day after castration.
