creators_name: Garg, Saurabh K
creators_name: Alam, Md Suhail
creators_name: Kishan, K V Radha
creators_name: Agrawal, Pushpa
type: article
datestamp: 2012-01-09 04:21:02
lastmod: 2012-01-09 04:21:02
metadata_visibility: show
title: Expression and characterization of alpha-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv.
ispublished: pub
subjects: QR
full_text_status: restricted
keywords: Mycobacterium tuberculosis; glgB; Glucan branching enzyme; Trypsin cleavage; ANS fluorescence; Redox-dependent protein conformation; Disulfide bond
note: Copyright of this article belongs to Elsevier Science.
abstract: Glycogen branching enzyme (GlgB, EC 2.4.1.18) catalyzes the third step of glycogen biosynthesis by the cleavage of an alpha-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new alpha-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of alpha-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 degrees C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.
date: 2007-02
date_type: published
publication: Protein expression and purification
volume: 51
number: 2
publisher: Elsevier Science
pagerange: 198-208
refereed: TRUE
issn: 1046-5928
official_url: http://www.sciencedirect.com/science/article/pii/S1046592806002555
related_url_url: http://www.sciencedirect.com/science/article/pii/S1046592806002555
related_url_type: pub
citation:   Garg, Saurabh K and Alam, Md Suhail and Kishan, K V Radha and Agrawal, Pushpa  (2007) Expression and characterization of alpha-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv.  Protein expression and purification, 51 (2).  pp. 198-208.  ISSN 1046-5928     
document_url: http://crdd.osdd.net/open/134/1/pushpa2007.pdf