The x-ray structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli
Abstract
Glutamine phosphoribosylpyrophosphate amidotransferases catalyze the first committed step in de novo purine biosynthesis. This thesis describes the x-ray crystallographic determination of the three dimensional structure of glutamine PRPP amidotransferase from E. coli to 2.5 A resolution by molecular replacement. The recently reported B. subtilis enzyme structure was used to devise a probe model. Whereas the B. subtilis enzyme contains an Fe4S4 cluster that may have a regulatory role and causes oxygen sensitivity, the E. coli enzyme does not contain iron. This metal cluster is not located in the active site and it has been suggested that it has a regulatory role since it causes the enzyme to be oxygen sensitive. Glutamine PRPP amidotransferase from Escherecia coli does not contain any iron and is not oxygen sensitive. The structure of the E. coli enzyme provided the opportunity to compare an Fe-containing and an Fe-free glutamine PRPP amidotransferase. The E. coli glutamine PRPP amidotransferase is an excellent target for mutational analysis of structure and function. Determination of the 3-D structure is a key aspect of this process. The B. subtilis enzyme x-ray structure showed the enzyme in its inhibited conformation, with eight AMP molecules bound per tetramer. The distance between the putative PRPP binding site and Cys1, which has been shown to bind glutamine analogs is about 15 A and a mechanism for NH$\sb3$ transfer is not obvious from the structure. A comparison to E. coli glutamine PRPP amidotransferase without AMP might give some information about the effects of binding this inhibitor. The structure of the E. coli enzyme reported here shows a similar separation of the two sites but only two AMP molecules are bound per tetramer and this partially inhibited state provides further insights into the inhibition mechanism.
Degree
Ph.D.
Advisors
Smith, Purdue University.
Subject Area
Molecules|Biophysics|Molecular biology
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