The PA4204 gene encodes a periplasmic gluconolactonase (PpgL) which is important for fitness of Pseudomonas aeruginosa.
In Pseudomonas aeruginosa, the PA4204 gene encodes a protein with a signal peptide and a COG2706 domain of the type present in 3-carboxy-cis,cis-muconate lactonizing enzymes. A molecular model based on the structure of the Escherichia coli YbhE phosphogluconate lactonizing enzyme shows that the enzyme has a beta-propeller ('doughnut') structure and a central active site comprising one histidine, one glutamic acid and two arginines. Inactivation of the P. aeruginosa PA4204 gene had profound phenotypic effects, resulting in slowly growing small colonies which frequently gave rise to larger colonies. The small colonies did not produce pyocyanin, produced reduced amounts of N-acylhomoserine lactones, and had extremely low levels of 2-alkyl-4-quinolones (AQs), while the larger colonies produced pyocyanin and higher amounts of AQs, including the pseudomonas quinolone signal (PQS), compared with the wild-type strain. Mutagenesis of His 182 in PA4204 resulted in the inability of this protein to restore pyocyanin production in the PA4204 isogenic mutant, suggesting that this enzyme may share an active site with other lactonizing enzymes. The protein with signal peptide was expressed as a His fusion in E. coli and purified. Two forms were observed, suggesting that the protein is translocated. The purified enzyme cleaved (S)-5-oxo-2-tetrahydrofurancarboxylic acid and d-glucono-delta-lactone, demonstrating lactonase activity. Decreased expression of the cytoplasmic phosphogluconolactonase gene (pgl) was observed in the small-colony mutant, and the mutant could not grow in the presence of mannitol or gluconate, suggesting functions in the detoxification of a gluconolactone and in sugar metabolism.
Tarighi
Saeed
S
Laboratory of Microbial Interactions, Department of Molecular and Cellular Interactions, Flanders Institute of Biotechnology (VIB), Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
Wei
Qing
Q
Laboratory of Microbial Interactions, Department of Molecular and Cellular Interactions, Flanders Institute of Biotechnology (VIB), Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
Cámara
Miguel
M
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK.
Williams
Paul
P
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK.
Fletcher
Matthew P
MP
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK.
Kajander
Tommi
T
Macromolecular X-ray Crystallography, Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Cornelis
Pierre
P
Laboratory of Microbial Interactions, Department of Molecular and Cellular Interactions, Flanders Institute of Biotechnology (VIB), Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005942:Gluconates / Q000378:metabolism; D007783:Lactones; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000201:enzymology*; D010641:Phenotype; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011710:Pyocyanine / Q000096:biosynthesis; D015363:Quinolones / Q000378:metabolism; D053038:Quorum Sensing; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C407944:2-heptyl-3-hydroxy-4-quinolone; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005942:Gluconates; D007783:Lactones; D015363:Quinolones; D011710:Pyocyanine; D002265:Carboxylic Ester Hydrolases; C020579:gluconolactonase; C010730:beta-glucono-1,5-lactone