Gene/Protein Disease Symptom Drug Enzyme Compound
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The biosynthesis of tryptophan in Mycobacterium tuberculosis is initiated by the transformation of chorismate to anthranilate, catalyzed by anthranilate synthase (TrpE/TrpG). Five additional enzymes are required to complete tryptophan biosynthesis. M. tuberculosis strains auxotrophic for tryptophan, an essential amino acid in the human diet, are avirulent. Thus, tryptophan synthesis in M. tuberculosis has been suggested as a potential drug target, and it has been reported that fluorinated anthranilate is lethal to the bacillus. Two mechanisms that could explain the cellular toxicity were tested: (1) the inhibition of tryptophan biosynthesis by a fluorinated intermediate or (2) formation of fluorotryptophan and its subsequent effects. Here, M. tuberculosis mc2 6230 cultures were treated with anthranilates fluorinated at positions 4, 5, and 6. These compounds inhibited bacterial growth on tryptophan-free media with 4-fluoroanthranilate being more potent than 5-fluoroanthranilate or 6-fluoroanthranilate. LC-MS based analysis of extracts from bacteria treated with these compounds did not reveal accumulation of any of the expected fluorinated intermediates in tryptophan synthesis. However, in all cases, significant levels of fluorotryptophan were readily observed, suggesting that the enzymes involved in the conversion of fluoro-anthranilate to fluorotryptophan were not being inhibited. Inclusion of tryptophan in cultures treated with the fluoro-anthranilates obviated the cellular toxicity. Bacterial growth was also inhibited in a dose-dependent manner by exposure to tryptophan substituted with fluorine at positions 5 or 6. Thus, the data suggest that fluorotryptophan rather than fluoro-anthranilate or intermediates in the synthesis of fluorotryptophan causes the inhibition of M. tuberculosis growth.
ACS Infect Dis 2019 01 11
PMID:Mechanism of Fluorinated Anthranilate-Induced Growth Inhibition in Mycobacterium tuberculosis. 3040 91

Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.
ACS Synth Biol 2019 04 19
PMID:Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase. 3086 44