title: Role of tryptophan residues in a class V chitinase from Nicotiana tabacum.
abstract: Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5-7 °C, while the mutations of Trp190 lowered stability only by 2-4 °C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)(4), in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the -2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the β-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite -2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115.
authors.affiliation: Department of Advanced Bioscience, Kinki University, Nakamachi, Nara, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Umemoto
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Henri
authors.initials: H
authors.lastname: Urpilainen
authors.forename: Takanori
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Numata
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine; D014364:Tryptophan; D002688:Chitinases; D000409:Alanine; D000117:Acetylglucosamine