Literature summary for 3.6.4.B7 extracted from

Topilina, N.I.; Novikova, O.; Stanger, M.; Banavali, N.K.; Belfort, M.
Post-translational environmental switch of RadA activity by extein-intein interactions in protein splicing (2015), Nucleic Acids Res., 43, 6631-6648 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene radA, recombinant expression in Escherichia coli strain BL21 Star (DE3), construction of the plasmid pMIG-RadAi, carrying the Pho RadA intein gene in foreign exteins, is accomplished by inserting the RadA intein sequence with short native exteins into ClaI and SphI sites between sequences coding for MBP and GFP in a pACYCDuet-1 plasmid backbone, splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state	Pyrococcus horikoshii

Cloned (Comment)

Organism

gene radA, recombinant expression in Escherichia coli strain BL21 Star (DE3), construction of the plasmid pMIG-RadAi, carrying the Pho RadA intein gene in foreign exteins, is accomplished by inserting the RadA intein sequence with short native exteins into ClaI and SphI sites between sequences coding for MBP and GFP in a pACYCDuet-1 plasmid backbone, splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state

Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
E354A	site-directed mutagenesis, mutant M9, matuation of a residue involved in ATPase function, and coordination of the nucleophilic water molecule	Pyrococcus horikoshii
E360A	site-directed mutagenesis, mutant M5, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii
E360A/R363A/E364A	site-directed mutagenesis, mutant M3, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii
E364A	site-directed mutagenesis, mutant M7, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii
E57A/K58A/R60A/E61A	site-directed mutagenesis, mutant M12, mutation of a residue that aisnot expected to be in proximity to intein catalytic residues	Pyrococcus horikoshii
E77A/K79A/E80A	site-directed mutagenesis, mutant M11, mutation of a residue that aisnot expected to be in proximity to intein catalytic residues	Pyrococcus horikoshii
Q465A	site-directed mutagenesis, mutant M10, matuation of a residue involved in ATPase function, and coordination of the nucleophilic water molecule	Pyrococcus horikoshii
R358A	site-directed mutagenesis, mutant M4, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii
R358A/E360A/R363A/E364A	site-directed mutagenesis, mutant M1, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii
R358A/R361A	site-directed mutagenesis, mutant M2, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii
R361A	site-directed mutagenesis, mutant M6, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii
R503A	site-directed mutagenesis, mutant M8, mutation of residues involved in exstein-intein interaction	Pyrococcus horikoshii

Inhibitors

Inhibitors	Comment	Organism	Structure
additional information	inhibitory RadA extein-intein interactions, overview. The catalytic residues of the intein are located on the extein-intein interface, revealing the possibility for 3D extein-intein interactions affecting intein catalysis	Pyrococcus horikoshii

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + H2O	Pyrococcus horikoshii	-	ADP + phosphate	-	?
ATP + H2O	Pyrococcus horikoshii OT-3	-	ADP + phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O58001	-	-
Pyrococcus horikoshii OT-3	O58001	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
additional information	splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. This splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies co-evolution of the parasitic intein and its host protein may provide another means of post-translational control. Extein-imposed inhibition of splicing can be modulated by solution environment. Post-translational regulation of RadA by superimposed mechanisms, native exteins as sensors that modulate intein splicing	Pyrococcus horikoshii

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	temperature-dependent structure transition andATPase activity of the PhoRadA protein and the precursor, overview	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + H2O	-	Pyrococcus horikoshii	ADP + phosphate	-	?
ATP + H2O	-	Pyrococcus horikoshii OT-3	ADP + phosphate	-	?

Subunits

Subunits	Comment	Organism
More	highly electrostatic secondary structure elements of the ATPase domain of RadA: helix 1 (D352-K367), loop 1 (R496-R503) and loop 2 (E524-D529)	Pyrococcus horikoshii

Synonyms

Synonyms	Comment	Organism
Pho RadA	-	Pyrococcus horikoshii
RadA	-	Pyrococcus horikoshii

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	temperature-dependent structure transition and ATPase activity of the PhoRadA protein and the precursor, overview	Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Pyrococcus horikoshii

General Information

General Information	Comment	Organism
additional information	splicing of RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. This splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide another means of post-translational control. Homology models for the RadA extein and intein are generated separately based on the closest templates for the extein: PDB ID 2ZUB, and for the intein: PDB ID 4E2T, molecular dynamics simulations, overview. The catalytic residues of the intein are located on the extein-intein interface, revealing the possibility for 3D extein-intein interactions affecting intein catalysis. Conserved residues of the intein C153, H245, H312, H323 and N324 are oriented toward the RadA exteins	Pyrococcus horikoshii