title: The role of active site aromatic residues in substrate degradation by the human chitotriosidase.
abstract: Human chitotriosidase (HCHT) is a glycoside hydrolase family 18 chitinase synthesized and secreted in human macrophages thought be an innate part of the human immune system. It consists of a catalytic domain with the (β/α)8 TIM barrel fold having a large area of solvent-exposed aromatic amino acids in the active site and an additional family 14 carbohydrate-binding module. To gain further insight into enzyme functionality, especially the effect of the active site aromatic residues, we expressed two variants with mutations in subsites on either side of the catalytic acid, subsite -3 (W31A) and +2 (W218A), and compared their catalytic properties on chitin and high molecular weight chitosans. Exchange of Trp to Ala in subsite -3 resulted in a 12-fold reduction in extent of degradation and a 20-fold reduction in kcat(app) on chitin, while the values are 5-fold and 10-fold for subsite +2. Moreover, aromatic residue mutation resulted in a decrease of the rate of chitosan degradation contrasting previous observations for bacterial family 18 chitinases. Interestingly, the presence of product polymers of 40 sugar moieties and higher starts to disappear already at 8% degradation for HCHT50-W31A. Such behavior contrast that of the wild type and HCHT-W218A and resembles the action of endo-nonprocessive chitinases.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway.
authors.forename: Kristine Bistrup
authors.initials: KB
authors.lastname: Eide
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway.
authors.forename: Linn Wilhelmsen
authors.initials: LW
authors.lastname: Stockinger
authors.affiliation: SINTEF Materials and Chemistry, Biotechnology and Nanomedicine Sector, Trondheim, Norway.
authors.forename: Anna Sofia
authors.initials: AS
authors.lastname: Lewin
authors.affiliation: SINTEF Materials and Chemistry, Biotechnology and Nanomedicine Sector, Trondheim, Norway.
authors.forename: Anne
authors.initials: A
authors.lastname: Tøndervik
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway.
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway. Electronic address: morten.sorlie@nmbu.no.
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
mesh_terms: D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002686:Chitin / Q000737:chemistry*; D048271:Chitosan / Q000737:chemistry*; D006596:Hexosaminidases / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008970:Molecular Weight; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D024322:Amino Acids, Aromatic; D002686:Chitin; D048271:Chitosan; D006596:Hexosaminidases; C086400:chitotriosidase
keywords: Glycoside hydrolases; aromatic residues; human chitotriosidase; processivity; recalcitrant polysaccharides