Using a molecular model and kinetic experiments in the presence of divalent cations to study the active site and catalysis of Pseudomonas aeruginosa phosphorylcholine phosphatase

2008 ◽  
Vol 1784 (12) ◽  
pp. 2038-2044 ◽  
Author(s):  
Paola R. Beassoni ◽  
Lisandro H. Otero ◽  
Angela T. Lisa ◽  
Carlos E. Domenech
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Molecular Model ◽  
Divalent Cations ◽  
Phosphorylcholine Phosphatase

scholarly journals Oligoribonuclease is the primary degradative enzyme for pGpG inPseudomonas aeruginosathat is required for cyclic-di-GMP turnover

2015 ◽  
Vol 112 (36) ◽  
pp. E5048-E5057 ◽  
Author(s):  
Mona W. Orr ◽  
Gregory P. Donaldson ◽  
Geoffrey B. Severin ◽  
Jingxin Wang ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Half Life ◽  
Biofilm Formation ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Capillary Action ◽  
Diguanylate Cyclases ◽  
Active Site Mutants ◽  
Degradative Enzyme

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A fromPseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆ornstrains ofP. aeruginosaPA14 for pGpG stability. The lysates from ∆ornshowed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆ornstrain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulatedpelpromoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆ornstrain in apel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆ornstrain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

scholarly journals Avian reovirus core protein μA expressed in Escherichia coli possesses both NTPase and RTPase activities

2007 ◽  
Vol 88 (6) ◽  
pp. 1797-1805 ◽  
Author(s):  
Yu Pin Su ◽  
Jui Huang Shien ◽  
Hung Jen Liu ◽  
Hsien Sheng Yin ◽  
Long Huw Lee
Keyword(s):  
Active Site ◽  
Rna Synthesis ◽  
Core Protein ◽  
Divalent Cations ◽  
Fusion Peptide ◽  
Enzymic Activity ◽  
Avian Reovirus ◽  
Phosphate Release ◽  
N Terminus ◽  
Rna Triphosphatase

Analysis of the amino acid sequence of core protein μA of avian reovirus has indicated that it may share similar functions to protein μ2 of mammalian reovirus. Since μ2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant μA ( μA) was designed and used to test these activities. μA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that  μA possessed NTPase activity that enabled the protein to hydrolyse the β–γ phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k cat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of  μA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein  μA. The activity of  μA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of  μA was achieved between pH 5.5 and 6.0. In addition,  μA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with  μA or K408A/K412A  μA indicated that  μA, but not K408A/K412A  μA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of  μA might be carried out at the same active site, and that protein μA could play important roles during viral RNA synthesis.

scholarly journals Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase

1992 ◽  
Vol 286 (1) ◽  
pp. 23-30 ◽  
Author(s):  
M F Hoylaerts ◽  
T Manes ◽  
J L Millán
Keyword(s):  
Germ Cell ◽  
Active Site ◽  
Molecular Model ◽  
Inhibition Mechanism ◽  
Side Group ◽  
Alkaline Phosphatases ◽  
Uncompetitive Inhibition ◽  
Guanidinium Group ◽  
Hydrolysis Of ◽  
Site Binding

Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.

scholarly journals Influence of the α-Methoxy Group on the Reaction of Temocillin with Pseudomonas aeruginosa PBP3 and CTX-M-14 β-Lactamase

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Michael D. Sacco ◽  
Kyle G. Kroeck ◽  
M. Trent Kemp ◽  
Xiujun Zhang ◽  
Logan D. Andrews ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Methoxy Group ◽  
Complex Structure ◽  
Antibacterial Properties ◽  
Multidrug Resistant ◽  
Binding Modes ◽  
Content Type ◽  
The Stability ◽  
M 14

ABSTRACT The prevalence of multidrug-resistant Pseudomonas aeruginosa has led to the reexamination of older “forgotten” drugs, such as temocillin, for their ability to combat resistant microbes. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The α-methoxy modification confers resistance to serine β-lactamases, yet temocillin is ineffective against P. aeruginosa growth. The origins of temocillin’s inferior antibacterial properties against P. aeruginosa have remained relatively unexplored. Here, we analyze the reaction kinetics, protein stability, and binding conformations of temocillin and ticarcillin with penicillin-binding protein 3 (PBP3), an essential PBP in P. aeruginosa. We show that the 6-α-methoxy group perturbs the stability of the PBP3 acyl-enzyme, which manifests in an elevated off-rate constant (koff) in biochemical assays comparing temocillin with ticarcillin. Complex crystal structures with PBP3 reveal similar binding modes of the two drugs but with important differences. Most notably, the 6-α-methoxy group disrupts a high-quality hydrogen bond with a conserved residue important for ligand binding while also being inserted into a crowded active site, possibly destabilizing the active site and enabling water molecule from bulk solvent to access and cleave the acyl-enzyme bond. This hypothesis is supported by the observation that the acyl-enzyme complex of temocillin has reduced thermal stability compared with ticarcillin. Furthermore, we explore temocillin’s mechanism of β-lactamase inhibition with a high-resolution complex structure of CTX-M-14 class A serine β-lactamase. The results suggest that the α-methoxy group prevents hydrolysis by locking the compound into an unexpected conformation that impedes access of the catalytic water to the acyl-enzyme adduct.

scholarly journals Phosphorylcholine Phosphatase: A Peculiar Enzyme of Pseudomonas aeruginosa

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Carlos Eduardo Domenech ◽  
Lisandro Horacio Otero ◽  
Paola Rita Beassoni ◽  
Angela Teresita Lisa
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Adaptive Response ◽  
Metal Ion ◽  
Coordination Geometry ◽  
Choline Metabolism ◽  
Inhibitory Site ◽  
Closed Structure ◽  
Phosphorylcholine Phosphatase ◽  
Hydrolysis Of

Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it is more effective at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+ induces at pH 5.0 a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. However, at pH 7.4, this effect is not observed because to the hydrolysis of the [Zn2+L2−1L20(H2O)2] complex, which causes a change from octahedral to tetrahedral in the metal coordination geometry. This enzyme is also present in P. fluorescens, P. putida, P. syringae, and other organisms. We have recently crystallized PchP and solved its structure.

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