Abstract 2250 The problematic intermediate that….isn't?

Proline is an essential precursor for protein synthesis. As the only secondary amino acid in proteins, it plays an important role in kinking and rigidifying polypeptide backbones. The pathway for synthesis of proline in most organisms begins with phosphorylation of L-glutamate to gamma-glutamyl phosphate (GP) by glutamate 5-kinase (ProB). GP is subsequently reduced to glutamyl semialdehyde by gamma-glutamyl phosphate reductase (ProA). A striking feature of this ancient pathway is that GP is unstable, rapidly cyclizing to 5-oxoproline. For decades, it has been assumed that channeling of GP between the active sites of ProB and ProA prevents cyclization. Despite the appeal of this idea, the experimental evidence is either not convincing or, in our hands, not reproducible. We have explored an alternative hypothesis inspired by the report that ProB-GFP and ProA-GFP localize to the poles in E. coli. Colocalization of ProB and ProA at the poles would increase the probability that GP would encounter a ProA active site before it had a chance to cyclize. However, we found that localization of ProB-GFP and ProA-GFP to the poles is an artifact of the fusion of the proteins to GFP, which can cause aggregation and localization to the poles due to nucleoid exclusion. We are currently reassessing the channeling hypothesis by testing whether alterations in the surfaces of ProB and ProA that would be expected to disrupt a physical interaction have adverse consequences in vivo and in vitro. This work was funded by the National Institute for General Medical Sciences.