Catalytic properties of key active site mutants of flavocytochrome P-450 BM3

1999 ◽  
Vol 27 (1) ◽  
pp. A44-A44 ◽  
Author(s):  
M.A. Noble ◽  
C.S. Miles ◽  
G.A. Reid ◽  
S.K. Chapman ◽  
A.W. Munro
Keyword(s):  
Active Site ◽  
Catalytic Properties ◽  
Active Site Mutants

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 A comparative study of class-D β-lactamases

1993 ◽  
Vol 292 (2) ◽  
pp. 555-562 ◽  
Author(s):  
P Ledent ◽  
X Raquet ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Gene Sequence ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Beta Lactamases ◽  
Class D ◽  
Burst Kinetics

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

Kinetic and Structural Analysis of Active Site Mutants of Monofunctional NAD-Dependent 5,10-Methylenetetrahydrofolate Dehydrogenase fromSaccharomyces cerevisiae†

Biochemistry ◽  
2005 ◽  
Vol 44 (39) ◽  
pp. 13163-13171 ◽  
Author(s):  
Wendi Wagner ◽  
Andrew P. Breksa ◽  
Arthur F. Monzingo ◽  
Dean R. Appling ◽  
Jon D. Robertus
Keyword(s):  
Structural Analysis ◽  
Active Site ◽  
Methylenetetrahydrofolate Dehydrogenase ◽  
Active Site Mutants

scholarly journals Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis

2002 ◽  
Vol 269 (3) ◽  
pp. 893-901 ◽  
Author(s):  
Evert Bokma ◽  
Henriëtte J. Rozeboom ◽  
Mark Sibbald ◽  
Bauke W. Dijkstra ◽  
Jaap J. Beintema
Keyword(s):  
Active Site ◽  
Hevea Brasiliensis ◽  
Rubber Tree ◽  
Active Site Mutants

scholarly journals Active site mutants of human secreted Group IIA Phospholipase A2 lacking hydrolytic activity retain their bactericidal effect

Biochimie ◽  
2012 ◽  
Vol 94 (1) ◽  
pp. 132-136 ◽  
Author(s):  
Lucimara Chioato ◽  
Elisangela Aparecida Aragão ◽  
Tatiana Lopes Ferreira ◽  
Richard J. Ward
Keyword(s):  
Phospholipase A2 ◽  
Active Site ◽  
Hydrolytic Activity ◽  
Bactericidal Effect ◽  
Active Site Mutants ◽  
Group Iia Phospholipase A2
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