Related Experiment Video
Updated: Mar 24, 2026
Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase
Published on: November 23, 2016
Substrate specificity and function of acetylpolyamine amidohydrolases from Pseudomonas aeruginosa
Andreas Krämer1, Jan Herzer2, Joerg Overhage2
1Department of Chemical Engineering and Biotechnology, University of Applied Sciences, Haardtring 100, 64295, Darmstadt, Germany.
Background:
Pseudomonas aeruginosa, a Gram-negative, aerobic coccobacillus bacterium is an opportunistic human pathogen and worldwide the fourth most common cause of hospital-acquired infections which are often high mortality such as ventilator-associated pneumoniae. The polyamine metabolism of P. aeruginosa and particularly the deacetylation of acetylpolyamines has been little studied up to now. Results with other bacterial pathogens e.g., Y. pestis suggest that polyamines may be involved in the formation of biofilms or confer resistance against certain antibiotics.
Results:
To elucidate the role of acetylpolyamines and their enzymatic deacetylation in more detail, all three putative acetylpolyamine amidohydrolases (APAHs) from P. aeruginosa have been expressed in enzymatic active form. The APAHs PA0321 and PA1409 are shown to be true polyamine deacetylases, whereas PA3774 is not able to deacetylate acetylated polyamines. Every APAH can hydrolyze trifluoroacetylated lysine-derivatives, but only PA1409 and much more efficiently PA3774 can also process the plain acetylated lysine substrate. P. aeruginosa is able to utilize acetylcadaverine and acetylputrescine as a carbon source under glucose starvation. If either the PA0321 or the PA1409 but not the PA3774 gene is disrupted, the growth of P. aeruginosa is reduced and delayed. In addition, we were able to show that the APAH inhibitors SAHA and SATFMK induce biofilm formation in both PA14 and PAO1 wildtype strains.
Conclusions:
P. aeruginosa has two functional APAHs, PA0321 and PA1409 which enable the utilization of acetylpolyamines for the metabolism of P. aeruginosa. In contrast, the physiological role of the predicted APAH, PA3774, remains to be elucidated. Its ability to deacetylate synthetic acetylated lysine substrates points to a protein deacetylation functionality with yet unknown substrates.
Insights
Pseudomonas aeruginosa utilizes acetylpolyamines as a carbon source via two key enzymes, PA0321 and PA1409. Disrupting these genes hinders bacterial growth, while inhibitors promote biofilm formation.
Area of Science:
- Microbiology
- Biochemistry
Background:
- Pseudomonas aeruginosa is a significant opportunistic pathogen causing hospital-acquired infections.
- Polyamine metabolism and acetylpolyamine deacetylation in P. aeruginosa are poorly understood.
- Polyamines may influence biofilm formation and antibiotic resistance in bacterial pathogens.
Purpose of the Study:
- To investigate the role of acetylpolyamine amidohydrolases (APAHs) in P. aeruginosa.
- To determine the specific deacetylation activities of P. aeruginosa APAHs.
- To understand the physiological impact of APAH function and inhibition on P. aeruginosa.
Main Methods:
- Expression and enzymatic activity assays of three putative APAHs (PA0321, PA1409, PA3774).
- Substrate specificity testing using acetylated lysine derivatives.
- Gene disruption experiments to assess growth phenotypes.
- Biofilm formation assays in the presence of APAH inhibitors (SAHA, SATFMK).
Main Results:
- PA0321 and PA1409 function as polyamine deacetylases; PA3774 does not deacetylate polyamines but processes acetylated lysine.
- P. aeruginosa can metabolize acetylcadaverine and acetylputrescine as carbon sources.
- Disruption of PA0321 or PA1409 significantly reduces and delays bacterial growth.
- APAH inhibitors SAHA and SATFMK induce biofilm formation in P. aeruginosa.
Conclusions:
- P. aeruginosa possesses two functional APAHs (PA0321, PA1409) crucial for acetylpolyamine metabolism.
- The exact physiological role of PA3774 requires further investigation, but it may be involved in protein deacetylation.
- Modulating APAH activity impacts P. aeruginosa growth and biofilm formation, suggesting therapeutic potential.
Related Concept Videos
01:24Phase II Reactions: Acetylation Reactions
The substrates for acetylation are typically drugs or their metabolites with an amino, sulfonamide, or hydrazine functional group. Acetylation can occur at several points in the drug molecule, including primary, secondary, and...
02:42Acidity of 1-Alkynes
The acidic strength of hydrocarbons follows the order: Alkynes > Alkenes > Alkanes. The strength of an acid is commonly expressed in units of pKa — the lower the pKa, the stronger the acid. Among the hydrocarbons, terminal alkynes have lower pKa values and are, therefore, more acidic. For example, the pKa values for ethane, ethene, and acetylene are 51, 44, and 25, respectively, as shown here.
01:19Amines to Amides: Acylation of Amines
Next, the second equivalent of amine serves as a Brønsted base and deprotonates the quaternary...
01:07Acid Halides to Amides: Aminolysis
In the first step of the aminolysis mechanism, the amine attacks the carbonyl carbon of the acyl chloride to form a tetrahedral intermediate. In the second step, the carbonyl group is re-formed with the elimination of a chloride...
01:18Indirect-Acting Cholinergic Agonists: Mechanism of Action
Reversible inhibitors like edrophonium bind to a specific part of the enzyme called the anionic catalytic site. They form noncovalent bonds, which means they are not strongly attached to the enzyme. This creates a temporary and less stable enzyme–inhibitor complex,...
01:21Basicity of Aliphatic Amines
To measure the basicity of amines, two conventions are generally used. The first defines Kb as the basicity constant for the deprotonation reaction of water by the amine, as presented in Figure 1. Conventionally, lower Kb indicates higher...