Title page for ETD etd-12022004-201028


Type of Document Dissertation
Author Burnette, Ryan Nelson
URN etd-12022004-201028
Title A Physiological, Biochemical and Structural Analysis of Inositol Polyphosphate 5-Phosphatases from Arabidopsis thaliana and Humans
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Gillaspy, Glenda E. Committee Chair
Kim, Sunyoung Committee Member
Larson, Timothy J. Committee Member
Luckhart, Shirley Committee Member
McDowell, John M. Committee Member
Tu, Zhijian Jake Committee Member
Keywords
  • 4
  • inositol (1
  • 5)-trisphosphate
  • inositol polyphosphate 5-phosphatase
  • Arabidopsis thaliana
  • Lowe syndrome
  • infrared spectroscopy
Date of Defense 2004-11-22
Availability unrestricted
Abstract
The complete role of inositol signaling in plants and humans is still elusive. The

plant Arabidopsis thaliana contains fifteen predicted inositol polyphosphate 5-

phosphatases (5PTases, E.C. 3.1.3.36) that have the potential to remove a 5-phosphate

from various inositol second messenger substrates. To examine the substrate specificity

of one of these Arabidopsis thaliana 5PTases (At5PTases), recombinant At5PTase1 was

obtained from a Drosophila melanogaster expression system and analyzed

biochemically. This analysis revealed that At5PTase1 has the ability to catalyze the

hydrolysis of four potential inositol second messenger substrates.

To determine whether At5PTase1 can be used to alter the signal transduction

pathway of the major drought-sensing hormone abscisic acid (ABA), plants ectopically

expressing At5PTase1 under the control of a constitutive promoter were characterized.

This characterization revealed that plants ectopically expressing At5PTase1 had an

altered response to ABA. These plants have stomata that are insensitive to ABA, and

have lower basal and ABA-induced inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] levels. In

addition, At5PTase1 mRNA and protein levels are transiently regulated by ABA. These

data strongly suggest that At5PTase1 can act as a signal terminator of ABA signal

transduction.

Like the Arabidopsis At5PTase1, a human 5PTase, Ocrl, has the ability to

catalyze the hydrolysis of a 5-phosphate from several inositol-containing substrates. The

loss of functional Ocrl protein results in a rare genetic disorder known as Lowe oculocerebrorenal syndrome. To gather information concerning the specificity

determinants of the Ocrl protein, a structure-function analysis of Ocrl was conducted

using a vibrational technique, difference Fourier transform infrared (FT-IR)

spectroscopy. Upon the introduction of Ins(1,4,5)P3 substrate, structural changes in

carboxylic acid and histidine residues were observed. The net result of changes in these

residues indicates that upon Ins(1,4,5)P3 introduction, a carboxylic acid-containing

residue is protonated, and a histidine residue is deprotonated. This interpretation supports

the idea that the deprotonation of the histidine residue is concomitant with the

coordination of a divalent cation upon Ins(1,4,5)P3 introduction. This work allows for the

proposal of a new model for the role of the active site histidine of OCRL.

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