Spinach thylakoid polyphenol oxidase: cloning, characterization, and relation to a putative protein kinase.

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)

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