Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2002
Publication Date: 12/30/2002
Citation: ZHANG,H., HUANG,X., FUKAMIZO,T., MUTHUKRISHNAN,S., KRAMER,K.J., SITE-DIRECTED MUTAGENESIS AND FUNCTIONAL ANALYSIS OF AN ACTIVE SITE TRYPTOPHAN OF INSECT CHITINASE, JOURNAL OF INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 32: 1477-1488. 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 constitutive over 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 mutagenesis and structure-activity 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 a specific aromatic amino acid contributes 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: Chitinase is an enzyme used by insects to degrade the structural polysaccharide, chitin, during the molting process. Tryptophan 145 (W145) of insect chitinase is a highly conserved residue found within a second conserved region of family 18 chitinases. It is located between aspartate 144 and glutamate 146, which are putative catalytic residues. The role of W145 in catalysis was investigated by site-directed mutagenesis. W145 was mutated to phenylalanine (F), tyrosine (Y), isoleucine (I), histidine (H), and glycine (G). Wild-type and mutant forms of were expressed in a baculovirus-insect cell line system. The chitinases were characterized by analyzing their catalytic activity and substrate or inhibitor binding properties. The wild-type chitinase was most active in the alkaline pH range. Several of the mutations resulted in a narrowing of the range of pH over which the enzyme hydrolyzed the polymeric substrate predominantly on the alkaline side of the pH optimum curve. The range was reduced by about pH unit for W145I and W145Y and by about 2 units for W145H and W145F. The W145G mutation was inactive. Therefore, the hydrophobicity of W145 appears to be critical for maintaining an abnormal pKa of a catalytic residue, which extends the activity further into the alkaline range. All of the mutant enzymes bound to chitin, suggesting that W145 was not essential for binding to chitin. The variations in kcat's among the mutated enzymes and the IC50 for the transition state inhibitor analog, allosamidin, indicate that W145 also influences formation of the transition state during catalysis.