Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-β-1,4-mannanase: mannosyl binding promoted at subsite -2 and demoted at subsite -3
Research output: Contribution to journal › Journal article › Research › peer-review
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 oligosaccharide 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.
Original language | English |
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Journal | Biochemistry |
Volume | 49 |
Issue number | 23 |
Pages (from-to) | 4884-4896 |
Number of pages | 13 |
ISSN | 0006-2960 |
DOIs | |
Publication status | Published - 2010 |
- Amino Acid Substitution, Binding Sites, Carbohydrate Sequence, Cellulomonas, Conserved Sequence, Crystallography, X-Ray, Hydrolysis, Mannose, Mannosidases, Mutagenesis, Site-Directed, Protein Binding, Protein Engineering, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity
Research areas
ID: 41923226