Title:Bromein, a Bromelain Inhibitor from Pineapple Stem: Structural and Functional Characteristics
Volume: 25
Issue: 9
Author(s): Ken-ichi Hatano*, Kenji Takahashi and Masaru Tanokura
Affiliation:
- Division of Molecular Science, Faculty of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515,Japan
Keywords:
Bromelain inhibitor, gene structure, inhibition mechanism, precursor processing, site-directed mutagenesis, tertiary
structure.
Abstract: Bromelain inhibitor, “bromein”, is a proteinase-inhibitor specific to the cysteine proteinase
bromelain from pineapple stem. In the stem, eight bromein isoforms are known to exist, and
each isoform has a short peptide (light chain) and a long one (heavy chain) with five disulfide
bonds. The three-dimensional structure of the sixth isoform (bromein-6) is composed of inhibitory
and stabilizing domains, and each domain contains a three-stranded antiparallel β-sheet. The genomic
sequence of a bromein precursor encodes three homologous bromein isoform domains, and
each isoform domain has a signal peptide, three interchain peptides between the light chain and
heavy chain, two interdomain peptides and a propeptide. Interestingly, at the protein level, bromein-
6 appears to share a similar folding and disulfide-bonding connectivity with Bowman-Birk serine
proteinase inhibitors and shows weak inhibition toward chymotrypsin and trypsin. However, no
significant similarity was found between them at the genomic level. This indicates that they have
evolved convergently to possess such a structural similarity. To identify the essential reactive site(s)
with bromelain, we investigated the inhibitory activity of 44 kinds of the single/double and insertion/
deletion mutants of bromein-6 towards stem bromelain. As a result, it was shown that both the
appropriate positioning and the complete side-chain structure of Leu10 in the light chain are absolutely
crucial for the inhibition, with an additional measure of importance for the preceding Pro9.
Bromein and stem bromelain coexist in the acidic vacuoles of the stem tissue, and one of the key
role of bromein appears to be the regulation of the bromelain activity.
