Kiwi protein inhibitor of pectin methylesterase - Amino-acid sequence and structural importance of two disulfide bridges

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid se quence of the pectin methylesterase inhibitor (PMEI) was determined by dire ct protein analysis. The sequence comprises 152 amino-acid residues, accoun ting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protei n has five cysteine residues but neither tryptophan nor methionine. Analysi s of fragments obtained after digestion of the protein alkylated without pr evious reduction identified two disulfide bridges connecting Cys9 with Cys1 8, and Cys74 with Cys114; Cys140 bears a free thiol group. A database searc h pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulf ide bridges, are conserved. This allows us to infer that also in the homolo gous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitu te a common structural motif. The comparison of the sequence of these inhib itors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inh ibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.