Site-Directed Mutagenesis of N5-Carboxyaminoimidazole Ribonucleotide Mutase (Class I PurE) in Bacillus anthracis

Abstract

Anthrax, the infection caused by the Gram-positive pathogen Bacillus anthracis (B. anthracis), is fatal if untreated, and some strains of B. anthracis have been found to be resistant to currently available antibiotics. The development of broad spectrum antibiotics is needed to treat the resistive strains. In antibiotic development, we have targeted B. anthracis Class I PurE enzyme (Ba PurE) as a unique and necessary enzyme in the de novo purine biosynthesis pathway, since the inactivation of this gene prevents B. anthraci growth in human serum, resulting in decreased bacterial proliferation. To identify inhibitors to $Ba$PurE, structural information on the substrate binding to its active site is needed. However, it is difficult to obtain crystals of Ba PurE with the substrate molecule in its binding site since upon binding to PurE, the substrate molecule is converted to the product molecule. An alternative approach is to create mutants of PurE that exhibit no enzymatic activity and do not convert the substrate to product, but still allow the substrate to bind to the active site. Then, the structure of mutant PurE with bound substrate can be obtained. We have identified a histidine residue at position 70 as the target of mutation to give an inactive enzyme. After successfully preparing the recombinant protein H70N, we have found that it exhibited no enzyme activity. This mutant will be useful in future experimentation to identify inhibitors of Ba PurE.