Breadcrumb

 
 

Expression, purification, crystallization and preliminary X-ray analysis of PR-AMP cyclohydrolase : a zinc binding enzyme from methanobacterium thermoautotrophicum

Title:

Expression, purification, crystallization and preliminary X-ray analysis of PR-AMP cyclohydrolase : a zinc binding enzyme from methanobacterium thermoautotrophicum

Boju, Lorena (2004) Expression, purification, crystallization and preliminary X-ray analysis of PR-AMP cyclohydrolase : a zinc binding enzyme from methanobacterium thermoautotrophicum. Masters thesis, Concordia University.

[img]
Preview
PDF - Accepted Version
3197Kb

Abstract

Histidine is an essential amino acid since humans and other higher eukaryotes lack the cellular biosynthetic machinery for its manufacture. Phosphoribosyl-AMP cyclohydrolase (PRA-CH, EC 3.5.4.19) is the third enzyme in the histidine biosynthetic pathway. PRA-CH catalyzes the hydrolysis of the N1-C6 bond of the purine substrate N1-(5 ' -phosphoribosyl)adenosine 5 ' -monophosphate (PR-AMP). The enzyme from Methanobacterium thermoautotrophicum , overexpressed in E. coli , was selected for crystallization trials as steps towards obtaining its three dimensional structure by X-ray diffraction. Initial attempts of purifying the recombinant hexa histidine tag protein were not successful. A new construct encoding for PRA-CH without the His-tag was made, overexpressed in E. coli and purified to homogeneity using heat treatment followed by one chromatographic step. Yields of 50-60 mg were obtained per liter of culture. The enzyme was shown by Dynamic Light Scattering studies to form a homodimer of 31 500 Da. Crystals of the protein were formed at an unusually low concentration of 2.1 mg/ml by the vapour-diffusion method using sodium acetate as precipitant. The presence of 50 mM Cd(SO 4 ) 2 in the reservoir solution was essential for the crystallization of the protein. Crystals appeared within two weeks at room temperature. They belong to the orthorhombic system, space group P2 1 2 1 2 1 , with cell dimensions a = 39.7Å, b = 54.3Å, c = 117.3Å, [Special characters omitted.] = [up down arrow] = [white square] = 90{493}. These crystals diffracted to 1.76Å at the synchrotron beamline. Several attempts to determine the 3-D structure of PR-AMP cyclohydrolase by multiwavelength anomalous dispersion were unsuccessful. Following completion of the thesis, however, the structure was eventually solved by single wavelength anomalous dispersion.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Boju, Lorena
Pagination:xiii, 72 leaves : ill. (some col.) ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry and Biochemistry
Date:2004
Thesis Supervisor(s):Cygler, Miroslaw and Turnbull, Joanne
ID Code:8075
Deposited By:Concordia University Libraries
Deposited On:18 Aug 2011 14:14
Last Modified:18 Aug 2011 15:48
Related URLs:
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Document Downloads

More statistics for this item...

Concordia University - Footer