Exploring the Coenzyme A Biosynthetic Pathway as Novel Antibiotic Target.

Abstract

Coenzyme A (CoA) is an essential molecule for all organisms. CoA is biosynthesized via five enzymatic steps from the metabolic precursor pantothenate. Phosphopantetheine, the product of the first three enzymatic steps, is ultimately attached to apo-acyl carrier proteins (ACP) to generate active, holo-ACP, which is reported to participate in up to 13% of all characterized biochemical reactions including essential steps in fatty acid metabolism. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of CoA biosynthesis. PPCS from Enterococcus faecalis and human were cloned, purified, and characterized. While PPCS from both species exhibited similar ordered Bi Uni Uni Bi Ping Pong mechanisms, significant differences in substrate selectivity were found between the bacterial and human PPCS homologues. E. faecalis PPCS was CTP-specific, while human PPCS could use both ATP and CTP in addition to being able to bind UTP and GTP. The CTP specificity of E. faecalis and other bacterial PPCS contrasted with the lack of nucleotide specificity of human PPCS suggests that selective inhibition of bacterial PPCS as a potential anti-bacterial should be feasible. A high throughput screen was conducted against a library of 41,000 small molecules and 11,000 natural product extracts to find novel inhibitors of bacterial PPCS. Several small molecules and natural product extracts were confirmed to be inhibitors of bacterial PPCS, and the inhibition mechanism for one inhibitor was determined. Phosphopantetheine adenylyltransferase (PPAT), the enzyme involved in the fourth step of CoA biosynthesis, was also studied, leveraging the technologies developed in the previous PPCS studies. PPAT from E. faecalis was cloned, purified, and characterized. E. faecalis PPAT was determined to have an ordered Bi Bi mechanism, in contrast to the random Bi Bi mechanism of the previously characterized E. coli PPAT. A high throughput screen to find inhibitors of PPAT was developed, but no specific inhibitors were found in the pilot screen.