The aspartate aminotransferase family in conifers:: biochemical analysis of a prokaryotic-type enzyme from maritime pine
The aspartate aminotransferase family in conifers:: biochemical analysis of a prokaryotic-type enzyme from maritime pine
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DOI：
10.1093/treephys/27.9.1283
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发表时间：
2007-09-01
期刊：
影响因子：
4
通讯作者：
Canovas, Francisco M.
中科院分区：
文献类型：
--
作者：
De La Torre, Fernando;Suarez, Maria Fernanda;Canovas, Francisco M.
植物天冬氨酸氨基转移酶(AAT，EC 2.6.1.1)在初级氮同化、还原当量的转移和亚细胞间碳氮库的交换中起着关键作用。以海松为实验模型，对针叶树AAT家族进行了研究。鉴定了胞质、线粒体和两种胞质同工酶(真核型和原核型)的基因，并比较了它们的氨基酸序列。真核型酶的一级结构非常保守，而原核型AAT的一级结构高度分化(同源性为15%)。这两种类型的天然AAT之间没有免疫交叉反应，证实了这些分子数据。在大肠杆菌中高效表达了成熟的原核型多肽，并对天然酶进行了纯化，得到了明显的同源性，并对其分子性质进行了测定。完全活性的重组全酶在50-60℃时显示出最高的催化活性，并且具有中等的热稳定性，在70℃孵育5-10分钟后，其活性仍保持在50%左右。5‘-磷酸吡哆醛的存在显著提高了酶的热稳定性。利用这些分子特征，建立了一种从松子叶中快速纯化这种原核型酶的方法。这一结果将为研究树木的天冬氨酸和氨基酸代谢提供参考。
Plant aspartate aminotransferase (AAT, EC 2.6.1.1) plays a key role in primary nitrogen assimilation, the transfer of reducing equivalents and the interchanges of carbon and nitrogen pools between subcellular compartments. We investigated the AAT family in conifers using maritime pine as the experimental model. Genes for cytosolic, mitochondrial and two plastidic isoenzymes (eukaryotic- and prokaryotic-types) were identified and their deduced amino acid sequences compared. The primary structure of the eukaryotic-type enzymes is quite well conserved, whereas the prokaryotic-type AAT is highly divergent (15% of identity). These molecular data were confirmed by the absence of immunological cross-reactivity between the two types of native AATs. The mature prokaryotic-type polypeptide was overexpressed in Escherichia coli, and the native enzyme was purified to apparent homogeneity and its molecular properties determined. The fully active recombinant holoenzyme showed highest catalytic activity at 50-60 degrees C and was moderately thermostable, retaining about 50% of its activity after incubation at 70 degrees C for 5-10 min. The presence of pyridoxal 5'-phosphate significantly increased the thermostability of the enzyme. These molecular characteristics were exploited to develop a rapid protocol for the purification of this prokaryotic-type enzyme from pine cotyledons. The results will be useful for Studying aspartate and amino acid metabolism in trees.