Bromein, a Bromelain Inhibitor from Pineapple Stem: Structural and Functional Characteristics

Area of Science:

• Biochemistry
• Plant Molecular Biology
• Enzymology

Background:

• Bromelain inhibitor (bromein) is a cysteine proteinase inhibitor found in pineapple stems.
• Bromein exists in eight isoforms, each with light and heavy chains linked by disulfide bonds.
• Bromein-6 shares structural similarities with Bowman-Birk inhibitors, suggesting convergent evolution.

Purpose of the Study:

• To identify the critical amino acid residues responsible for bromein's inhibitory activity against stem bromelain.
• To elucidate the functional role of specific domains and residues within bromein.

Main Methods:

• Site-directed mutagenesis was used to create 44 single/double and insertion/deletion mutants of bromein-6.
• Inhibitory activity of bromein-6 mutants against stem bromelain was assessed.
• Structural and genomic comparisons with other proteinase inhibitors were performed.

Main Results:

• The Leu10 residue in the light chain is essential for bromein's inhibitory function.
• The Pro9 residue preceding Leu10 also plays a significant role in inhibition.
• Bromein and stem bromelain are co-localized in stem vacuoles, indicating a regulatory relationship.

Conclusions:

• Specific residues, particularly Leu10 and Pro9, are crucial for bromein's interaction with and inhibition of stem bromelain.
• Bromein likely functions to regulate stem bromelain activity within pineapple stem vacuoles.
• Convergent evolution explains the structural similarities between bromein and unrelated serine proteinase inhibitors.