The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis
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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 980643
- Report Number(s):
- BNL-93561-2010-JA; JMOBAK; TRN: US201015%%2028
- Journal Information:
- Journal of Molecular Biology, Vol. 385; ISSN 0022-2836
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
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