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
(2010) In Biochemistry 49(23). p.4884-4896- Abstract
- 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... (More)
- 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. (Less)
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https://lup.lub.lu.se/record/1632289
- author
- Hekmat, Omid LU ; Lo Leggio, Leila ; Rosengren, Anna LU ; Kamarauskaite, Jurate ; Kolenová, Katarina LU and Stålbrand, Henrik LU
- organization
- publishing date
- 2010
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochemistry
- volume
- 49
- issue
- 23
- pages
- 4884 - 4896
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- wos:000278452300020
- scopus:77953243269
- pmid:20426480
- ISSN
- 0006-2960
- DOI
- 10.1021/bi100097f
- language
- English
- LU publication?
- yes
- id
- a98fb723-b0e0-4618-a5c8-03c176c3fe6f (old id 1632289)
- date added to LUP
- 2016-04-01 10:42:14
- date last changed
- 2022-03-04 22:01:34
@article{a98fb723-b0e0-4618-a5c8-03c176c3fe6f,
abstract = {{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.}},
author = {{Hekmat, Omid and Lo Leggio, Leila and Rosengren, Anna and Kamarauskaite, Jurate and Kolenová, Katarina and Stålbrand, Henrik}},
issn = {{0006-2960}},
language = {{eng}},
number = {{23}},
pages = {{4884--4896}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Biochemistry}},
title = {{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}},
url = {{http://dx.doi.org/10.1021/bi100097f}},
doi = {{10.1021/bi100097f}},
volume = {{49}},
year = {{2010}},
}