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Expression, purification, crystallization and preliminary crystallographic analysis of a GH20 β‐ N ‐acetylglucosaminidase from the marine bacterium Vibrio harveyi
Author(s) -
Meekrathok Piyanat,
Bürger Marco,
Porfetye Arthur T.,
Vetter Ingrid R.,
Suginta Wipa
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x1500415x
Subject(s) - crystallization , dimer , chemistry , crystallography , glycoside hydrolase , substrate (aquarium) , mutant , vibrio harveyi , wild type , hydrolase , trimer , sodium acetate , stereochemistry , hydrolysis , bacteria , vibrio , enzyme , biochemistry , biology , organic chemistry , ecology , gene , genetics
Vibrio harveyi β‐ N ‐acetylglucosaminidase ( Vh GlcNAcase) is a new member of the GH20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with N ‐acetylglucosamine (GlcNAc) monomers as the final products. In this study, the crystallization and preliminary crystallographic data of wild‐type Vh GlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild‐type Vh GlcNAcase were grown in 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while crystals of the D437A mutant were obtained in 0.1 M bis‐tris pH 7.5, 0.1 M sodium acetate, 20% PEG 3350. X‐ray data from the wild‐type and the mutant crystals were collected at a synchrotron‐radiation light source and were complete to a resolution of 2.5 Å. All crystals were composed of the same type of dimer, with the substrate N , N ′‐diacetylglucosamine (GlcNAc 2 or diNAG) used for soaking was cleaved by the active enzyme, leaving only a single GlcNAc molecule bound to the protein.