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A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis

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Abstract

Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 Å resolution. AtChiC was found to adopt an (β/α)8 fold with a small insertion domain composed of an α-helix and a five-stranded β-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.

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Abbreviations

AtChiC:

Class V chitinase from Arabidopsis thaliana

NtChiV:

Class V chitinase from Nicotiana tabacum

SmChiB:

Family GH-18 chitinase B from Serratia marcescens

GlcNAc:

2-Acetamido-2-deoxy-d-glucopyranose

(GlcNAc) n :

β-1,4-Linked oligosaccharide of GlcNAc with a polymerization degree of n

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

HPLC:

High performance liquid chromatography

MALDI-TOF-MS:

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

ABA:

Abscisic acid

JA:

Jasmonic acid

GA:

Gibberellic acid

RMS:

Root mean square

EST:

Expression sequence tag

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Acknowledgments

This work was partly supported by a grant to O.L. from the North Ostrobothnia Regional Fund of the Finnish Cultural Foundation. The authors thank the beam-line staffs at BL-17A of KEK (Ibaraki, Japan) for technical assistance during data collection, and Hideko Inanaga of AIST for technical assistance on protein crystallography.

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Authors and Affiliations

  1. Department of Advanced Bioscience, Kinki University, 3327–204 Nakamachi, Nara, 631-8505, Japan

    Takayuki Ohnuma, Mamiko Mizuhara & Tamo Fukamizo

  2. Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566, Japan

    Tomoyuki Numata & Takuo Osawa

  3. Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen, Denmark

    Karen Skriver

  4. Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland

    Outi Lampela & André H. Juffer

Authors
  1. Takayuki Ohnuma
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  2. Tomoyuki Numata
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  3. Takuo Osawa
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  4. Mamiko Mizuhara
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  5. Outi Lampela
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  6. André H. Juffer
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  7. Karen Skriver
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  8. Tamo Fukamizo
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Corresponding authors

Correspondence to Tomoyuki Numata or Tamo Fukamizo.

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Ohnuma, T., Numata, T., Osawa, T. et al. A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis. Planta 234, 123–137 (2011). https://doi.org/10.1007/s00425-011-1390-3

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  • DOI: https://doi.org/10.1007/s00425-011-1390-3

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