Characterization of the indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa PAO1

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in the lungs of individuals with cystic fibrosis. It is intrinsically resistant to many antibiotics, and resistance is emerging rapidly to those drugs that currently remain efficacious. Therefore, there is a pressing need to identify new anti-pseudomonal drug targets. To this end, we have characterized the P. aeruginosa indole-3-glycerol phosphate synthase (PaIGPS). PaIGPS catalyzes the fifth reaction in the synthesis of tryptophan from chorismate--a reaction that is absent in mammals. PaIGPS was expressed heterologously in Escherichia coli, and purified with high yields. The purified enzyme is active over a broad pH range and has the highest turnover number of any characterized IGPS (k (cat) = 11.1 ± 0.1 s(-1)). These properties are likely to make PaIGPS useful in coupled assays for other enzymes in tryptophan biosynthesis. We have also shown that deleting the gene for PaIGPS reduces the fitness of P. aeruginosa strain PAO1 in synthetic cystic fibrosis sputum (relative fitness, W = 0.89 ± 0.02, P = 0.001). This suggests that de novo tryptophan biosynthesis may play a role in the establishment and maintenance of P. aeruginosa infections, and therefore that PaIGPS is a potential target for the development of new anti-pseudomonal drugs.

Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation

“…4B), and similar to the rate of CdRP turnover with EcIGPS (0.29 ± 0.03 s -1 ). The previously reported kcat for PaIGPS is 11 s -1 (17) and for monofunctional EcIGPS 3.2 s -1 (22).…”

“…The tryptophan biosynthesis enzyme IGPS has been heavily studied due to its potential significance for medical and industrial applications. In this work, we have probed the structure and mechanism of IGPS from P. aeruginosa, since it has the highest reported turnover number of all IGPS enzymes characterized to date (17).…”

“…This has made it useful in coupled activity assays of other Trp biosynthetic enzymes, such as evolved HisA mutants with PRAI activity (18). We also showed that deleting trpC from P. aeruginosa incurred a fitness cost of ~11% in synthetic cystic fibrosis sputum medium (17), suggesting that inhibition of IGPS may be a useful strategy for helping to clear P. aeruginosa infections from the lungs of cystic fibrosis patients, where tryptophan levels can be undetectably low (19).…”

“…We previously reported the initial characterization of IGPS from the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa (17). This enzyme, PaIGPS, has a higher kcat value than the other IGPSs that have been characterized (kcat = 11 s -1 ).…”

Desalting DNA by Drop Dialysis Increases Library Size upon Transformation

“…This mimics the construction of an error-prone PCR library. A 338-bp DNA fragment was ligated with the 4.3-kb expression vector pLAB101 11) after each had been digested with restriction enzymes NdeI and SpeI (both New England Biolabs, Ipswich, MA, USA). Three ligation reactions were performed in a total volume of 60 mL per reaction.…”