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Publication Abstract from PubMed

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M(-1)s(-1), with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E-64, solved at 1.73 and 1.98A, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E-64 and ananain explains the high inhibitory efficiency of E-64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.

Determination of the crystal structure and substrate specificity of ananain.,Yongqing T, Wilmann PG, Pan J, West ML, Brown TJ, Mynott T, Pike RN, Wijeyewickrema LC Biochimie. 2019 Jul 12. pii: S0300-9084(19)30202-0. doi:, 10.1016/j.biochi.2019.07.011. PMID:31306685^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.