2x2y | pdb_00002x2y

Cellulomonas fimi endo-beta-1,4-mannanase double mutant

spinqualitylabelsall models 2x2y, resolution 2.35Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2x2y is a 2 chain structure with sequence from Cellulomonas fimi. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.35Å Ligands: FMT, MG Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function Q9XCV5_CELFI Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 A resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of (18)O-labeled products obtained using H(2)(18)O as a solvent. The results obtained with oligosacharide substrates show that although the catalytic efficiency (k(cat)/K(m)) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K(m) decreased approximately 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme. Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-beta-1,4-mannanase: mannosyl binding promoted at subsite -2 and demoted at subsite -3 .,Hekmat O, Lo Leggio L, Rosengren A, Kamarauskaite J, Kolenova K, Stalbrand H Biochemistry. 2010 Jun 15;49(23):4884-96. PMID:20426480 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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