Mechanistic Analysis of the Aspartate Aminotransferase Active Site Mutants—Y70F, K258A, and R292D

1987 ◽  
pp. 59-67 ◽  
Author(s):  
Jack F. Kirsch ◽  
Wayne L. Finlayson ◽  
Michael D. Toney ◽  
Ciaran N. Cronin
Biochemistry ◽  
1991 ◽  
Vol 30 (7) ◽  
pp. 1980-1985 ◽  
Author(s):  
Avis T. Danishefsky ◽  
James J. Onnufer ◽  
Gregory A. Petsko ◽  
Dagmar Ringe

1974 ◽  
Vol 249 (20) ◽  
pp. 6684-6692
Author(s):  
Yoshimasa Morino ◽  
Abdalla Mohamed Osman ◽  
Mitsuhiro Okamoto

2015 ◽  
Vol 112 (36) ◽  
pp. E5048-E5057 ◽  
Author(s):  
Mona W. Orr ◽  
Gregory P. Donaldson ◽  
Geoffrey B. Severin ◽  
Jingxin Wang ◽  
Herman O. Sintim ◽  
...  

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.


Biochemistry ◽  
2005 ◽  
Vol 44 (39) ◽  
pp. 13163-13171 ◽  
Author(s):  
Wendi Wagner ◽  
Andrew P. Breksa ◽  
Arthur F. Monzingo ◽  
Dean R. Appling ◽  
Jon D. Robertus

2002 ◽  
Vol 269 (3) ◽  
pp. 893-901 ◽  
Author(s):  
Evert Bokma ◽  
Henriëtte J. Rozeboom ◽  
Mark Sibbald ◽  
Bauke W. Dijkstra ◽  
Jaap J. Beintema

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