6MIS

Native ananain in complex with E-64

Summary for 6MIS

• Entry DOI: 10.2210/pdb6mis/pdb
• Descriptor: Ananain, N-[N-[1-HYDROXYCARBOXYETHYL-CARBONYL]LEUCYLAMINO-BUTYL]-GUANIDINE (3 entities in total)
• Functional Keywords: pineapple cysteine protease e-64 inhibitor complex, plant protein, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Ananas comosus (Pineapple)
• Total number of polymer chains: 2
• Total formula weight: 47451.85

Authors

Yongqing, T.,Wilmann, P.G.,Pike, R.N.,Wijeyewickrema, L.C. (deposition date: 2018-09-20, release date: 2018-10-03, Last modification date: 2024-11-20)

Primary citation

Yongqing, T.,Wilmann, P.G.,Pan, J.,West, M.L.,Brown, T.J.,Mynott, T.,Pike, R.N.,Wijeyewickrema, L.C.
Determination of the crystal structure and substrate specificity of ananain.
Biochimie, 166:194-202, 2019

PubMed 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 k/K value of 1.7 x 106 Ms, 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.

PubMed: 31306685
DOI: 10.1016/j.biochi.2019.07.011

Experimental method

X-RAY DIFFRACTION (1.98 Å)