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Determination of the crystal structure and substrate specificity of ananain.
Biochimie. 2019 Nov; 166:194-202.B

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.

Authors+Show Affiliations

Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia; Anatara Lifesciences Ltd., Brisbane, Australia.Monash Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Melbourne, Australia.Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia.Anatara Lifesciences Ltd., Brisbane, Australia.Anatara Lifesciences Ltd., Brisbane, Australia.Anatara Lifesciences Ltd., Brisbane, Australia.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.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.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31306685

Citation

Yongqing, Tang, et al. "Determination of the Crystal Structure and Substrate Specificity of Ananain." Biochimie, vol. 166, 2019, pp. 194-202.
Yongqing T, Wilmann PG, Pan J, et al. Determination of the crystal structure and substrate specificity of ananain. Biochimie. 2019;166:194-202.
Yongqing, T., Wilmann, P. G., Pan, J., West, M. L., Brown, T. J., Mynott, T., Pike, R. N., & Wijeyewickrema, L. C. (2019). Determination of the crystal structure and substrate specificity of ananain. Biochimie, 166, 194-202. https://doi.org/10.1016/j.biochi.2019.07.011
Yongqing T, et al. Determination of the Crystal Structure and Substrate Specificity of Ananain. Biochimie. 2019;166:194-202. PubMed PMID: 31306685.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determination of the crystal structure and substrate specificity of ananain. AU - Yongqing,Tang, AU - Wilmann,Pascal G, AU - Pan,Jing, AU - West,Michael L, AU - Brown,Tracey J, AU - Mynott,Tracey, AU - Pike,Robert N, AU - Wijeyewickrema,Lakshmi C, Y1 - 2019/07/12/ PY - 2019/03/18/received PY - 2019/07/10/accepted PY - 2019/7/16/pubmed PY - 2019/12/31/medline PY - 2019/7/16/entrez KW - Ananain KW - Cysteine protease KW - Substrate specificity SP - 194 EP - 202 JF - Biochimie JO - Biochimie VL - 166 N2 - 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. SN - 1638-6183 UR - https://www.unboundmedicine.com/medline/citation/31306685/Determination_of_the_crystal_structure_and_substrate_specificity_of_ananain_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0300-9084(19)30202-0 DB - PRIME DP - Unbound Medicine ER -