Abstract
Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition.
Highlights
Cysteine proteinases are involved in specific processing or more general degradation of proteins in a wide variety of organisms, including viruses, fungi, plants, and animals [1]
Bromelain is known as cysteine proteinases in the stem and fruit of Ananas comosas, while the inhibitors exist only in the stem and have been classified into eight isoforms based on their amino acid sequences [10]
The presence of inhibitory fractions has been confirmed in pineapple stem [13], and the amino acid sequence of the seventh bromein was the first sequence determined among the inhibitory fractions [4]
Summary
Cysteine proteinases are involved in specific processing or more general degradation of proteins in a wide variety of organisms, including viruses, fungi, plants, and animals [1]. We constructed a recombinant single-chain sixth bromein with the interchain peptide (bromein-6R, Fig. 1B) and revealed that it shows almost the same inhibitory activity and secondary structure as bromein-6N [20]. Bromein-6R is processed between the light chain and interchain peptide by stem bromelain, the target proteinase [19].
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