Biochemical and molecular analysis of an Arabidopsis Nudix hydrolase.

摘要

Scope of Study. The family of Nudix hydrolases, termed as house cleaning enzymes, play vital role in pyridine nucleotide homeostasis by catalyzing the hydrolysis of a variety of nucleoside diphosphate derivatives. The major goal of this study is to characterize the function of Nudt7, a cytosolic nudix hydrolase enzyme, in Arabidopsis thaliana. NUDT7 transcript levels accumulate rapidly (within 15mins) in response to biotic and abiotic stresses. Recombinant NUDT7 protein hydrolyzes NADH and ADPR. Knockout mutant of AtNUDT7 (nudt7) exhibits multiple pleiotropic phenotypes such as reduced size, increased resistance to pathogens, increased ROS and NADH levels. The current project involves utilization of genetic and biochemical approaches to understand the in vivo function of Nudt7. The genomic approach includes whole-genome Affymetrix Gene Chip analysis performed to identify the genes that were differentially expressed between the wild type and nudt7 knock out plants. The biochemical approaches involve utilization of two different enzyme cycling assays and HPLC to measure and compare the in planta levels of pyridine nucleotides and ADP-ribose in Col-0 and nudt7. The significant alteration in gene expression and alteration of PN balance caused by knocking out of a single gene, suggest that the altered pyridine nucleotide balance might have led to the gene expression changes. However, further work has to be done to prove it.