Jespersen H M, Kjaersgård I V, Ostergaard L, Welinder K G
Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, Denmark.
Biochem J. 1997 Sep 1;326 ( Pt 2)(Pt 2):305-10. doi: 10.1042/bj3260305.
Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thaliana ascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event.
抗坏血酸过氧化物酶是一类血红素蛋白，能有效地清除植物细胞质和叶绿体中的过氧化氢。数据库分析检索到52个编码拟南芥抗坏血酸过氧化物酶的表达序列标签。对非冗余克隆进行全序列测序后发现，除了之前在拟南芥中描述的两种细胞质类型外，还有三种新类型。对所有植物抗坏血酸过氧化物酶的序列数据进行分析后得出以下分类：两种细胞质可溶性抗坏血酸过氧化物酶，分别命名为cs1和cs2；三种细胞质膜结合抗坏血酸过氧化物酶，即cm1，通过C端跨膜区段与微体结合，以及cm2和cm3，二者位置均未知；两种具有N端转运序列的叶绿体抗坏血酸过氧化物酶，即基质抗坏血酸过氧化物酶（chs）和类囊体结合抗坏血酸过氧化物酶，后者具有C端跨膜区段，命名为cht。豌豆抗坏血酸过氧化物酶保守残基模式与晶体结构的进一步比较表明，活性位点残基是保守的，除cm2和cm3类型外，与还原底物相互作用的三个肽段相似。细胞质类型中第175位的苯丙氨酸变为叶绿体类型中的色氨酸，这可能解释了叶绿体抗坏血酸过氧化物酶比细胞质抗坏血酸过氧化物酶具有更高抗坏血酸特异性的原因。参与同型二聚体亚基相互作用的残基仅在cs1、cs2和cm1类型中保守。在豌豆抗坏血酸过氧化物酶中观察到的近端阳离子（K+）结合位点似乎是保守的。此外，cm1、cm2、cm3、chs和cht抗坏血酸过氧化物酶含有第43位的天冬氨酸、第57位的天冬酰胺和第59位的丝氨酸，表明存在一个远端单价阳离子位点。这些数据支持了当今过氧化物酶是由早期基因复制事件进化而来的假说。
