Key message

A new Tau class GST gene was cloned from Pinus brutia Ten. cDNA sequence was analysed for conserved sequences. Substrate specificity, optimum pH, and temperature values of the recombinant PbGST Tau enzyme were determined.

Abstract

Tau class glutathione S-transferases (GSTs) are essential enzymes for detoxification in plants. To date, a lot of the members of this family have been characterized from different plants but the studies on the conifers are very scarce. This study investigates for the first time molecular cloning and biochemical characterization of a Tau class GST gene (PbGST Tau) from Pinus brutia Ten. The full length PbGST Tau ORF was 687 bp having a molecular mass of 27.37 kDa. Catalytic and ligand binding sites of PbGST Tau are well conserved and shared maximum identity with Pinus tabulaeformis GST Tau. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ECA) as substrates exhibited a K_m of 3.66 mM and 0.3 mM, respectively. PbGST Tau enzyme had an optimum activity at pH 6.0 and 8.0 when CDNB and ECA were used as substrate, respectively. The highest activity was measured at 25 °C. Through enzyme assays, phylogenetic analysis and structural modelling, we provide a detailed characterization of the PbGST Tau gene and the enzyme. This study is going to provide new insights into the phylogenetic and biochemical analysis of GST family in conifers.

中文翻译：

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松属植物Tau类谷胱甘肽S-转移酶的分子克隆和生化特性分析十

关键信息

从黑松10克隆了一个新的Tau类GST基因。分析cDNA序列的保守序列。确定了重组Pb GST Tau酶的底物特异性，最适pH和温度值。

抽象

Tau类谷胱甘肽S转移酶（GST）是植物中排毒的必需酶。迄今为止，该家族的许多成员已从不同的植物中鉴定出特征，但是对针叶树的研究非常匮乏。这项研究首次调查了来自十大松树（Pinus brutia Ten ）的Tau类GST基因（Pb GST Tau）的分子克隆和生化特性。全长Pb GST Tau ORF为687 bp，分子量为27.37 kDa。Pb GST Tau的催化和配体结合位点保存完好，与油松具有最大的同一性GST头 关于作为底物的1-氯-2，4-二硝基苯（CDNB）和乙炔酸（ECA）的动力学分析显示，K _m分别为3.66 mM和0.3 mM。当CDNB和ECA分别用作底物时，Pb GST Tau酶在pH 6.0和8.0时具有最佳活性。在25°C下测得最高活性。通过酶分析，系统发育分析和结构建模，我们提供了Pb GST Tau基因和酶的详细表征。这项研究将为针叶树中GST家族的系统发育和生化分析提供新的见解。