SPINACH THYLAKOID POLYPHENOL OXIDASE - CLONING, CHARACTERIZATION, ANDRELATION 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 sequ ence, of which the first 15 were identical to a sequence reported [Gal , A., Hermann, R. G., Lottspeich, F., & Ohad, I. (1999) FEES Lett. 298 ,; 33-35] for a protein kinase with specificity toward the photosystem II light-harvesting complex (LHC-P). 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 hyd rophobic 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 p rotein kinase motifs are absent, as are sequences characteristic of AT P-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 b egins a C-terminal domain containing 7 predicted beta-strands which, b y analogy with Hc, may form an antiparallel beta-barrel. We conclude t hat 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]. In view of its lumenal location and primary sequence, it is unlikely to be a serine/threonine protein kinase.