Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

Marx, Ute C., Korsinczky, Michael L. J., Schirra, Horst Joachim, Jones, Alun, Condie, Barrie, Otvos, Laszlo and Craik, David J. (2003) Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide. Journal of Biological Chemistry, 278 24: 21782-21789. doi:10.1074/jbc.M212996200

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Author Marx, Ute C.
Korsinczky, Michael L. J.
Schirra, Horst Joachim
Jones, Alun
Condie, Barrie
Otvos, Laszlo
Craik, David J.
Title Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
Publication date 2003-06-12
Sub-type Article (original research)
DOI 10.1074/jbc.M212996200
Open Access Status File (Publisher version)
Volume 278
Issue 24
Start page 21782
End page 21789
Total pages 8
Place of publication Bethesda
Publisher The American Society for Biochemistry amd Molecular Biology c
Collection year 2003
Language eng
Subject C1
250302 Biological and Medical Chemistry
780105 Biological sciences
Abstract The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[ 6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI1[6,5] is similar to9:1 regardless of whether trypsin is incubated with SFTI-1[ 6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[ 6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[ 6,5] revealed high heterogeneity, and multiple species of SFTI-1[ 6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an isoaspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.
Keyword Biochemistry & Molecular Biology
Serine-protease
Substrate-specificity
Plant Cyclotides
Knotted Proteins
Reactive-site
Seeds
Purification
Binding
Domain
Family
Q-Index Code C1

 
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Created: Wed, 15 Aug 2007, 02:48:27 EST