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

Research output: Contribution to journalArticle

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.

Details

Authors
  • Omid Hekmat
  • Leila Lo Leggio
  • Anna Rosengren
  • Jurate Kamarauskaite
  • Katarina Kolenová
  • Henrik Stålbrand
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology
Original languageEnglish
Pages (from-to)4884-4896
JournalBiochemistry
Volume49
Issue number23
Publication statusPublished - 2010
Publication categoryResearch
Peer-reviewedYes

Related research output

Rosengren, A., 2014, Department of Chemistry, Lund University. 180 p.

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