The different pH optima and substrate specificities of extracellular and vacuolar invertases from plants are determined by a single amino-acid substitution
The different pH optima and substrate specificities of extracellular and vacuolar invertases from plants are determined by a single amino-acid substitution
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DOI：
10.1046/j.1365-313x.1999.00628.x
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发表时间：
1999-12-01
期刊：
影响因子：
7.2
通讯作者：
Roitsch, T
中科院分区：
文献类型：
--
作者：
Goetz, M;Roitsch, T
不同的植物转化酶同工酶在保守序列WEC-P/V-D box上只有一个氨基酸的差异。在胞外转化酶序列的这个序列基序中存在一个脯氨酸残基，而在液泡转化酶序列的同一位置上发现了一个缬氨酸。这种显著差异的作用是通过定点突变将红色藜胞外转化酶CIN1的脯氨酸残基替换为Valine残基来研究的。突变基因在蔗糖酶缺陷型酿酒酵母中异源表达。单一氨基酸的差异是蔗糖酶同工酶最适pH和底物专一性两种酶性质的分子基础。WEC-P/V-D盒中的一个脯氨酸决定了胞外转化酶与在该位置有valine残基的空泡转化酶相比，更酸性的pH最适和更高的棉籽糖裂解率。
Different plant invertase isoenzymes are characterized by a single amino-acid difference in a conserved sequence, the WEC-P/V-D box. A proline residue is present in this sequence motif of extracellular invertase sequences, whereas a valine is found at the same position of vacuolar invertase sequences. The role of this distinct difference was studied by substituting the proline residue of extracellular invertase CIN1 from Chenopodium rubrum with a valine residue, by site-directed mutagenesis. The mutated gene was heterologously expressed in an invertase-deficient Saccharomyces cerevisiae strain. The single amino-acid difference was shown to be the molecular basis for two enzymatic properties of invertase isoenzymes, for both the pH optimum and the substrate specificity. A proline in the WEC-P/V-D box determines the more acidic pH optimum and the higher cleavage rate of raffinose of extracellular invertases, compared to vacuolar invertases that have a valine residue at this position.