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United States Department of Agriculture

Agricultural Research Service

Title: Site-Directed Mutagenesis and Functional Analysis of Active Site Acidic Amino Acid Residues D142, D144, and E146 in Manduca Sexta (Tobacco Hornworm) Chitinase

Authors
item Lu, Yimin - KANSAS STATE UNIVERSITY
item Muthukrishnan, Subbaratnam - KANSAS STATE UNIVERSITY
item Kramer, Karl

Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 9, 2002
Publication Date: November 30, 2002
Citation: LU,Y., MUTHUKRISHNAN,S., KRAMER,K.J., SITE-DIRECTED MUTAGENESIS AND FUNCTIONAL ANALYSIS OF ACTIVE SITE ACIDIC AMINO ACID RESIDUES D142, D144, AND E146 IN MANDUCA SEXTA (TOBACCO HORNWORM) CHITINASE, JOURNAL OF INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 32: 1369-1382. 2002.

Interpretive Summary: Agricultural crops worldwide suffer from a vast array of insect pests that cause severe yield losses. One of the strategies to increase plant tolerance to insect pests is the expression of proteins with insecticidal activity. In prior research we showed that an insect molting enzyme chitinase acts as a biopesticide in transgenic plants where it disrupts insect gut physiology. Together with scientists at Kansas State University we conducted experiments to help identify and characterize some of the amino acids that are essential for enzymatic and insecticidal activities. The data reveal which and how specific acidic amino acids contribute to the enzyme's mechanism of action. The results obtained provide useful information about the enzymatic and biopesticidal functions in plants and also structure-function relationships. The findings will add to the knowledge base for hydrolytic enzyme mechanisms and facilitate a more effective application of the insect chitinase gene for the control of insect pests. The long-term goal of this research is to improve the physical, chemical and kinetic properties of this biopesticide, including enzymatic activity, stability, and insecticidal activity.

Technical Abstract: Chitinases are glycosyl hydrolases that catalyze the hydrolysis of b-(1, 4)-glycosidic bonds in chitin. From amino acid sequence comparisons of glycosyl hydrolases, two conserved regions were identified. The second of these regions contains three very highly conserved acidic amino acid residues, D142, D144 and E146, that are probably active site residues. The functional roles of these three residues were investigated using site-directed mutagenesis for their substitutions to other amino acids. Six mutant proteins were produced. The proteins were purified by anion-exchange chromatography, after which their physical, kinetic, and substrate binding properties were determined. Circular dichroism spectra were similar to that of the wild-type protein, indicating that the presence of mutations did not change the overall secondary structures. E146 was found to be required for enzymatic activity because mutants E146Q and E146D Dwere devoid of activity. D144E retained most of the enzymatic activity, bu D144N lost nearly 90%. There was a shift in the pH optimum from alkaline pH to acidic pH for mutants D142N and D144E with minimal losses of activity. The pH-activity profile for the D142E mutation resembled that of the wild-type enzyme except activity in the neutral and acidic range was lower. All of the mutant proteins bound to chitin. Therefore, none of these acidic residues was essential for substrate binding. The results indicated that E146 functions as an acid/base catalyst in the hydrolytic mechanism and D144 as an electrostatic stabilizer of the positively charged transition state, whereas D142 probably influences the pKa values of D144 and E146.

Last Modified: 9/21/2014
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