Related Experiment Video
Updated: May 23, 2025

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants
Published on: May 4, 2018
Pseudomonas aeruginosa acyl-CoA dehydrogenases and structure-guided inversion of their substrate specificity
Meng Wang1, Prasanthi Medarametla1,2, Thales Kronenberger2,3,4
1Department of Biochemistry, Tennis Court Road, Cambridge, UK.
Abstract:
Fatty acids are a primary source of carbon for Pseudomonas aeruginosa (PA) in the airways of people with cystic fibrosis (CF). Here, we use tandem mass-tag proteomics to analyse the protein expression profile of a CF clinical isolate grown on different fatty acids. Two fatty acyl-CoA dehydrogenases (designated FadE1 and FadE2) are strongly induced during growth on fatty acids. FadE1 displays a strong preference for long-chain acyl-CoAs, whereas FadE2 exclusively utilizes medium-chain acyl-CoAs. Structural analysis of the enzymes enables us to identify residues comprising the substrate selectivity filter in each. Engineering these residues enables us to invert the substrate specificity of each enzyme. Mutants in fadE1 displayed impaired virulence in an infection model, and decreased growth on long chain fatty acids. The unique features of the substrate binding pocket enable us to identify an inhibitor that is differentially active against FadE1 and FadE2.
Related Concept Videos
SN2 Reaction: Stereochemistry
If the substrate is an achiral molecule at the α-carbon, the inversion of configuration is not...
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Enzymes
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Induced-fit Model
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
Ligand Binding and Linkage
SN1 Reaction: Stereochemistry
In the first step of an SN1 reaction, the bond between the electrophilic carbon and the leaving group ionizes to generate the carbocation intermediate. The second step of the mechanism is the nucleophilic attack.
In the formed carbocation, the positively charged carbon is sp2 hybridized with a trigonal planar geometry. As all the three substituents lie on the same plane, a plane of symmetry for the...

