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Title: The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis

Abstract

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with ?-galactosidase activity (Escherichia coli LacZ), ?-glucuronidase activity (Homo sapiens GusB), and ?-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-?-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural ?-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-?-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
980643
Report Number(s):
BNL-93561-2010-JA
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US201015%%2028
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Journal of Molecular Biology
Additional Journal Information:
Journal Volume: 385; Journal ID: ISSN 0022-2836
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; ARCHITECTURE; CLASSIFICATION; ENZYMES; GALACTOSIDASE; GLUCOSAMINE; GLUCOSE; GLUCURONIDASE; GLYCOSIDES; HYDROLASES; HYDROLYSIS; INTERACTIONS; MUTANTS; NITROGEN; RESIDUES; RESOLUTION; SPECIFICITY; SUBSTRATES; national synchrotron light source

Citation Formats

Van Bueren, A, Ghinet, M, Gregg, K, Fleury, A, Brzezinski, R, and Boraston, A. The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis. United States: N. p., 2009. Web. doi:10.1016/j.jmb.2008.10.031.
Van Bueren, A, Ghinet, M, Gregg, K, Fleury, A, Brzezinski, R, & Boraston, A. The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis. United States. https://doi.org/10.1016/j.jmb.2008.10.031
Van Bueren, A, Ghinet, M, Gregg, K, Fleury, A, Brzezinski, R, and Boraston, A. Thu . "The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis". United States. https://doi.org/10.1016/j.jmb.2008.10.031.
@article{osti_980643,
title = {The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis},
author = {Van Bueren, A and Ghinet, M and Gregg, K and Fleury, A and Brzezinski, R and Boraston, A},
abstractNote = {Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with ?-galactosidase activity (Escherichia coli LacZ), ?-glucuronidase activity (Homo sapiens GusB), and ?-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-?-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural ?-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-?-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.},
doi = {10.1016/j.jmb.2008.10.031},
url = {https://www.osti.gov/biblio/980643}, journal = {Journal of Molecular Biology},
issn = {0022-2836},
number = ,
volume = 385,
place = {United States},
year = {2009},
month = {1}
}