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Biochimie. 2019 Jul 12. pii: S0300-9084(19)30202-0. doi: 10.1016/j.biochi.2019.07.011. [Epub ahead of print]

Determination of the crystal structure and substrate specificity of ananain.

Author information

1
Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia; Anatara Lifesciences Ltd., Brisbane, Australia.
2
Monash Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Melbourne, Australia.
3
Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia.
4
Anatara Lifesciences Ltd., Brisbane, Australia.
5
Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia; ARC Centre of Excellence for Advanced Molecular Imaging. Melbourne, Australia. Electronic address: r.pike@latrobe.edu.au.
6
Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia; ARC Centre of Excellence for Advanced Molecular Imaging. Melbourne, Australia. Electronic address: l.wijeyewickrema@latrobe.edu.au.

Abstract

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-1s-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 1.98 Å, 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.

KEYWORDS:

Ananain; Cysteine protease; Substrate specificity

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