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
| Autor: | Henrik Stålbrand, Jurate Kamarauskaite, Anna Rosengren, Katarina Kolenova, Omid Hekmat, Leila Lo Leggio |
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| Rok vydání: | 2010 |
| Předmět: |
Stereochemistry
Crystallography X-Ray Protein Engineering Biochemistry Substrate Specificity Hydrolysis Hydrolase Mannosidases Glycoside hydrolase Enzyme kinetics Conserved Sequence Cellulomonas chemistry.chemical_classification Cellvibrio japonicus Binding Sites biology Hydrogen bond biology.organism_classification Affinities Enzyme chemistry Amino Acid Substitution Carbohydrate Sequence Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Mutagenesis Site-Directed Mannose Protein Binding |
| Zdroj: | Biochemistry. 49(23) |
| ISSN: | 1520-4995 |
| Popis: | 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 angstrom 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 O-18-labeled products obtained using (H2O)-O-18 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 similar to 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme. |
| Databáze: | OpenAIRE |
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