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Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation

Paper ID Volume ID Publish Year Pages File Format Full-Text
24244 43507 2009 8 PDF Available
Title
Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation
Abstract

The topology of Alg8, the proposed catalytic subunit of the alginate polymerase, was assessed using PhoA and LacZ fusion protein analysis. This analysis suggested that the periplasmic loop comprises only three amino acid residues with the adjacent transmembrane helices at positions 361–387 and 393–416. Accordingly, the extended cytosolic loop could be located at positions 71–361 and was proposed to contain important catalytic residues. Further experimental evidence for this cytosolic domain was obtained by independently demonstrating this protein region as purified soluble protein domain. The soluble protein domain was identified by MALDI-TOF/MS and presumably represents the cytosolic catalytic domain of Alg8. Site-directed mutagenesis of 11 conserved residues in the cytosolic loop showed that D-188/D-190 (DXD motif), D-295/D-296 (acid–base catalysts) and K-297 were each essential for in vivo polymerase activity, whereas D-179/D-181 (DXD motif), C-244, R-263, D-279, and E-282 were not directly involved in the polymerisation reaction. The role of these amino acid residues with respect to the catalysed alginate polymerisation reaction was discussed with the aid of the recently developed structural model of Alg8.

Keywords
Alginate biosynthesis; Pseudomonas aeruginosa; Alginate; Alginate polymerase
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Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 140, Issues 3–4, 25 March 2009, Pages 176–183
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
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