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
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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. / Hekmat, Omid; Lo Leggio, Leila; Rosengren, Anna; Kamarauskaite, Jurate; Kolenova, Katarina; Stålbrand, Henrik.
In: Biochemistry, Vol. 49, No. 23, 2010, p. 4884-4896.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-β-1,4-mannanase
T2 - mannosyl binding promoted at subsite -2 and demoted at subsite -3
AU - Hekmat, Omid
AU - Lo Leggio, Leila
AU - Rosengren, Anna
AU - Kamarauskaite, Jurate
AU - Kolenova, Katarina
AU - Stålbrand, Henrik
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - Amino Acid Substitution
KW - Binding Sites
KW - Carbohydrate Sequence
KW - Cellulomonas
KW - Conserved Sequence
KW - Crystallography, X-Ray
KW - Hydrolysis
KW - Mannose
KW - Mannosidases
KW - Mutagenesis, Site-Directed
KW - Protein Binding
KW - Protein Engineering
KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
KW - Substrate Specificity
U2 - 10.1021/bi100097f
DO - 10.1021/bi100097f
M3 - Journal article
C2 - 20426480
VL - 49
SP - 4884
EP - 4896
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 23
ER -
ID: 41923226