Investigation on Binding and Catalytic Activity of Adenylate Isopentenyltransferase from Humulus lupulus to Diadenosine Polyphosphates
Date Issued
2010
Date
2010
Author(s)
Chen, Feng-Yuan
Abstract
Diadenosine polyphosphates (ApnAs) are important signaling molecules. They are regarded as intracellular signaling molecules and involved in stress response, DNA replication and repair, and apoptosis. Moreover, out of cells, they play roles in cardiovascular system, where proliferation of vascular smooth muscle and vascular tone are affected by them, and in neuroendocrine system as one neurotransmitter. Although the occurrence of ApnAs has been demonstrated in various bacteria, fungi and animals, identification of these compounds in plants has not been reported so far. However, there are enzymes to synthesize and degrade in plant for those molecules. Those enzymes provide indirect evidence for the existence of ApnA in plant. Levels of ApnAs seem to be precisely controlled by a set of different enzymes, although their biological targets of regulation are still unclear. Data from isothermal titration calorimetry (ITC) showed that, ApnAs displayed different binding affinities to HlAIPT, and one of them is surprisingly with higher binding affinities than that of the original substrate (ATP) with an order of Ap5A > ATP > Ap4A > Ap6A > Ap3A. Compared with that of ATP, specific activities of HlAIPT to ApnA were further measured, and the order becomes ATP > Ap6A > Ap4A > Ap5A > Ap3A. These results implicated that the length of polyphosphate chain in ApnA would affect the binding and activity of HlAIPT to them. Therefore, mutations to investigate the effect of residues involving in binding of ApnA with different length based on modeling result on the activity of HlAIPT to ApnA were made. Results showed that the specific activity of R271A mutant to Ap3A and Ap5A were both increased, implying that R271A might decrease the steric hindrance and could therefore cause the increase of enzyme activity to ApnAs. Results from our study and the fact that plant AIPTs (HlAIPT, AtIPT1, AtIPT3, AtIPT5, and AtIPT8) and plant Ap4A hydrolases (AtNUDT28/AtNUDX26 and AtNUDT29/AtNUDX27) are all located in the chloroplast suggested that the existence of Ap4A in chloroplast is very possible and accordingly could be a natural substrate for AIPT. Our study here is a starting point to ask the role of ApnA in plants and might open avenues for another promising perspective of regulatory function of unusual nucleotide derivatives.
Subjects
ApnA
the existence of ApnA in plant
HlAIPT
binding affinity
specific activity
point mutation
Type
thesis
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