Hind G, Marshak D R, Coughlan S J
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Biochemistry. 1995 Jun 27;34(25):8157-64. doi: 10.1021/bi00025a022.
A 64-kDa protein was purified from an octyl glucoside/cholate extract of spinach thylakoids. N-Terminal analysis yielded 23 residues of sequence, of which the first 15 were identical to a sequence reported [Gal, A., Herrmann, R. G., Lottspeich, F., & Ohad, I. (1992) FEBS Lett. 298, 33-35] for a protein kinase with specificity toward the photosystem II light-harvesting complex (LHC-II). We report the complete sequence of this 64-kDa protein, deduced from cDNA clones. The transit peptide has a chloroplast import signal at the N-terminus and a C-terminal hydrophobic span bounded by basic amino acids that predicts localization of the protein to the thylakoid lumen. The mature protein sequence is about 50% identical to several polyphenol oxidases (PPOs). Canonical protein kinase motifs are absent, as are sequences characteristic of ATP-binding sites. The mature protein resembles arthropodan hemocyanin (Hc), possessing three major domains. The N-terminal domain is rich in cysteine residues and predicted alpha-helices. The central domain has a conserved motif, N-terminal to a presumptive Cu-A site, that is not found in tyrosinases or Hc and is proposed as the provider of a third imidazole ligand to Cu-A. An unusual 13-residue, glutamine-rich link begins a C-terminal domain containing 7 predicted beta-strands which, by analogy with Hc, may form an antiparallel beta-barrel. We conclude that this 64-kDa polypeptide is a lumenal PPO and the precursor of a 42.5-kDa PPO form described previously [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].(ABSTRACT TRUNCATED AT 250 WORDS)
从菠菜类囊体的辛基葡糖苷/胆酸盐提取物中纯化出一种64 kDa的蛋白质。N端分析得到了23个氨基酸残基的序列，其中前15个与报道的[Gal, A., Herrmann, R. G., Lottspeich, F., & Ohad, I. (1992) FEBS Lett. 298, 33 - 35]对光系统II捕光复合体（LHC-II）具有特异性的蛋白激酶的序列相同。我们报道了从cDNA克隆推导得到的这种64 kDa蛋白质的完整序列。转运肽在N端有叶绿体导入信号，C端有一个由碱性氨基酸界定的疏水片段，预测该蛋白质定位于类囊体腔。成熟蛋白质序列与几种多酚氧化酶（PPO）约50%相同。不存在典型的蛋白激酶基序，也没有ATP结合位点的特征序列。成熟蛋白质类似于节肢动物血蓝蛋白（Hc），具有三个主要结构域。N端结构域富含半胱氨酸残基和预测的α螺旋。中央结构域在假定的Cu-A位点的N端有一个保守基序，该基序在酪氨酸酶或Hc中未发现，被认为是为Cu-A提供第三个咪唑配体的提供者。一个不寻常的富含谷氨酰胺的13个残基的连接段开始了一个C端结构域，该结构域包含7个预测的β链，类似于Hc，可能形成一个反平行的β桶。我们得出结论，这种64 kDa的多肽是一种类囊体腔PPO，是先前描述的42.5 kDa PPO形式的前体[Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977 - 984]。（摘要截短至250字）
