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J. Biol. Chem., Vol. 283, Issue 42, 28287-28296, October 17, 2008

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An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila^*

Mohan Chandra Joshi¹, Animesh Sharma^¶, Sashi Kant, Ajanta Birah^||, Gorakh Prasad Gupta^||, Sharik R. Khan, Rakesh Bhatnagar, and Nirupama Banerjee²

From the International Center for Genetic Engineering and Biotechnology, New Delhi 110067, India, ^¶Fuzzylife, Open Source Bioinformatics Education, New Delhi 110091, India, ^||Division of Entomology, Indian Agricultural Research Institute, New Delhi 110012, India, and School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC₅₀ of 3.6 µg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed -chitin binding activity with K_d 0.64 µM and B_max 4.68 µmol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an 8-fold increase of K_d in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

Received for publication, June 9, 2008 , and in revised form, July 23, 2008.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY184491.

^* This work was supported in part by the Department of Biotechnology, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a senior research fellowship from Council of Scientific and Industrial Research, India.

² To whom correspondence should be addressed: International Center for Genetic Engineering and Biotechnology, New Delhi, India 110067. Fax: 91-11-26162316.

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