Tài liệu Báo cáo khoa học: C-Terminal extension of a plant cysteine protease modulates proteolytic

C-Terminal extension of a plant cysteine protease

modulates proteolytic activity through a partial inhibitory

mechanism

Sruti Dutta, Debi Choudhury, Jiban K. Dattagupta and Sampa Biswas

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India

Keywords

C-terminal extension; cysteine proteases;

modulation of proteolytic activity;

papain-like; thermostable

Correspondence

S. Biswas, Crystallography and Molecular

Biology Division, Saha Institute of Nuclear

Physics, 1 ⁄ AF Bidhannagar, Kolkata

700 064, India

Fax: +91 332 337 4637

Tel: +91 332 337 5345

E-mail: [email protected]

(Received 14 March 2011, revised 16 May

2011, accepted 22 June 2011)

doi:10.1111/j.1742-4658.2011.08221.x

The amino acid sequence of ervatamin-C, a thermostable cysteine protease

from a tropical plant, revealed an additional 24-amino-acid extension at its

C-terminus (CT). The role of this extension peptide in zymogen activation,

catalytic activity, folding and stability of the protease is reported. For this

study, we expressed two recombinant forms of the protease in Escherichia

coli, one retaining the CT-extension and the other with it truncated. The

enzyme with the extension shows autocatalytic zymogen activation at a

higher pH of 8.0, whereas deletion of the extension results in a more active

form of the enzyme. This CT-extension was not found to be cleaved during

autocatalysis or by limited proteolysis by different external proteases.

Molecular modeling and simulation studies revealed that the CT-extension

blocks some of the substrate-binding unprimed subsites including the speci￾ficity-determining subsite (S2) of the enzyme and thereby partially occludes

accessibility of the substrates to the active site, which also corroborates the

experimental observations. The CT-extension in the model structure shows

tight packing with the catalytic domain of the enzyme, mediated by strong

hydrophobic and H-bond interactions, thus restricting accessibility of its

cleavage sites to the protease itself or to the external proteases. Kinetic

stability analyses (T50 and t1 ⁄ 2) and refolding experiments show similar

thermal stability and refolding efficiency for both forms. These data suggest

that the CT-extension has an inhibitory role in the proteolytic activity of

ervatamin-C but does not have a major role either in stabilizing the enzyme

or in its folding mechanism.

Structured digital abstract

l ErvC cleaves ErvC by protease assay (View interaction)

l trypsin cleaves ErvC by protease assay (View interaction)

Introduction

Papain-like cysteine proteases (EC 3.4.22) from plant

sources are of industrial and biotechnological impor￾tance because these enzymes are better suited to various

industrial processes [1]. A cysteine protease is expressed

as an inactive precursor in a pre-proenzyme form

which contains a signal peptide (pre-), an inhibitory

Abbreviations

CT, C-terminal; E-64, 1-[L-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane; Erv-C, ervatamin-C; pNA, p-nitroanilide; rmErv-C+CT,

recombinant mature ervatamin-C with C-terminal extension; rmErv-CDCT, recombinant mature ervatamin-C without C-terminal extension;

rproErv-C+CT, recombinant proervatamin-C with C-terminal extension; rproErv-CDCT, recombinant proervatamin-C without C-terminal

extension.

3012 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS