Crystal structure and mode of action of a class V chitinase from Nicotiana tabacum
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A class V chitinase from Nicotiana tabacum (NtChiV) with amino acid sequence similar to that of Serratia marcescens chitinase B (SmChiB) was expressed in E. coli and purified to homogeneity. When N-acetylglucosamine oligosaccharides [(NAG)n] were hydrolyzed by the purified NtChiV, the second glycosidic linkage from the non-reducing end was predominantly hydrolyzed in a manner similar to that of SmChiB. NtChiV was shown to hydrolyze partially N-acetylated chitosan non-processively, whereas SmChiB hydrolyzes the same substrate processively. The crystal structure of NtChiV was determined by the single-wavelength anomalous dispersion method at 1.2 Å resolution. The protein adopts a classical (β/α)8-barrel fold (residues 1–233 and 303–348) with an insertion of a small (α + β) domain (residues 234–302). This is the first crystal structure of a plant class V chitinase. The crystal structure of the inactive mutant NtChiV E115Q complexed with (NAG)4 was also solved and exhibited a linear conformation of the bound oligosaccharide occupying −2, +1, +2, and +3 subsites. The complex structure corresponds to an initial state of (NAG)4 binding, which is proposed to be converted into a bent conformation through sliding of the +1, +2, and +3 sugar units to −1, +1, and +2 subsites. Although NtChiV is similar to SmChiB, the chitin-binding domain is present in the C-terminus of the latter, but not in the former. Aromatic amino acid residues found in the substrate binding cleft of SmChiB, including Trp97, are substituted with aliphatic residues in NtChiV. These structural differences appear to be responsible for NtChiV being a non-processive enzyme.
KeywordsNicotiana tabacum Class V chitinase Crystal structure Mode of hydrolysis Binding mode
The authors thank the beam-line staffs at BL-5A, BL-17A, NW12A, and NE3A of KEK (Ibaraki, Japan) for technical assistance during data collection, and Dr. Karl J. Kramer, USDA-ARS, Center for Grain and Animal Health Research (Manhattan, Kansas) for his reviewing the manuscript. Thanks are also due to Hideko Inanaga of AIST for technical assistance and Dr. Masahiro Tamoi of Kinki University for kindly providing the first strand cDNA of tobacco.
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