Kiwi protein inhibitor of pectin methylesterase - Amino-acid sequence and structural importance of two disulfide bridges
Kiwi protein inhibitor of pectin methylesterase - Amino-acid sequence and structural importance of two disulfide bridges
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DOI：
10.1046/j.1432-1327.2000.01510.x
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发表时间：
2000-07-01
期刊：
影响因子：
--
通讯作者：
Giovane, A
中科院分区：
文献类型：
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作者：
Camardella, L;Carratore, V;Giovane, A
从猕猴桃（Actinidia chinensis）果实中分离出一种蛋白质，可作为植物果胶甲酯酶的强力抑制剂。果胶甲酯酶抑制剂 (PMEI) 的完整氨基酸序列通过直接蛋白质分析确定。该序列包含 152 个氨基酸残基，分子量为 16 277 Da。远紫外圆二色光谱表明二级结构中主要是α螺旋构象。该蛋白质有五个半胱氨酸残基，但既没有色氨酸也没有蛋氨酸。对未经还原的烷基化蛋白质消化后获得的片段进行分析，发现有两个二硫键连接 Cys9 与 Cys18、Cys74 与 Cys114； Cys140 带有游离硫醇基团。数据库搜索指出 PMEI 和植物蔗糖酶抑制剂之间的相似性。特别是，PMEI 中涉及二硫键的四个 Cys 残基是保守的。这使我们能够推断，在仅通过cDNA测序推导出一级结构的同源蛋白质中，那些半胱氨酸残基参与两个二硫键，并构成共同的结构基序。这些抑制剂的序列比较证实了一类具有中等但显着序列保守性的新型蛋白质的存在，其中包括作为糖代谢酶抑制剂的植物蛋白，并且可能参与植物发育的各个步骤。
A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.