Literature summary for 7.4.2.8 extracted from

Halder, P.K.; Roy, C.; Datta, S.
Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa (2018), Proteins, 87, 276-288 .

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3) cells	Pseudomonas aeruginosa
gene pscN, recombinant expression of His-tagged enzyme PscN in Escherichia coli strain BL21(DE3), coexpression with untagged regulator protein PscL as insoluble complex	Pseudomonas aeruginosa

Protein Variants

Protein Variants	Comment	Organism
additional information	crosslinking of the ATPase-regulator complex with 2% glutaraldehyde at 27°C for 5 min in 20 mM phosphate, pH 8.0, and 100 mM NaCl, PscN-PscL structure modelling, overview	Pseudomonas aeruginosa

Inhibitors

Inhibitors	Comment	Organism	Structure
PscL	PscL downregulates the enzyme activity up to 80% when mixed with the enzyme PscN in a 1:2 ratio (PscN:PscL); UniProt ID A0A0H2Z9T1, PscL interacts with PscN and inhibits its ATPase activity. PscL inhibits PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). PscN-PscL structure modelling, overview	Pseudomonas aeruginosa

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.93	-	ATP	pH 7.8, 37°C	Pseudomonas aeruginosa
0.93	-	ATP	at pH 7.8 and 37°C	Pseudomonas aeruginosa

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ATP + H2O	Pseudomonas aeruginosa	-	ADP + phosphate	-	?
ATP + H2O + cellular protein[side 1]	Pseudomonas aeruginosa	-	ADP + phosphate + cellular protein[side 2]	-	?
ATP + H2O + cellular protein[side 1]	Pseudomonas aeruginosa UCBPP-PA14	-	ADP + phosphate + cellular protein[side 2]	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas aeruginosa	A0A0H2Z9F1	-	-
Pseudomonas aeruginosa	O30538	-	-
Pseudomonas aeruginosa UCBPP-PA14	A0A0H2Z9F1	-	-

Purification (Commentary)

Purification (Comment)	Organism
Ni-NTA column chromatography and Superdex 200 gel filtration	Pseudomonas aeruginosa
refolded recombinant His-tagged enzyme PscN by nickel affinity chromatography, and untagged regulator PscL by dialysis and gel filtration	Pseudomonas aeruginosa

Renatured (Commentary)

Renatured (Comment)	Organism
insoluble PcsN-PcsL aggregate from pellet by treatment with unfolding buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 6 M guanidine hydrochloride for 30 min. For proper refolding, five times in volume refolding buffer, containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% glycerol, is added and left for overnight at 4°C on a stirrer rotor. Insoluble protein aggregates are removed by centrifugation and the supernatant containing soluble proteins is subjected to dialysis in dialysis buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% glycerol, to remove guanidine hydrochloride	Pseudomonas aeruginosa

Renatured (Comment)

Organism

insoluble PcsN-PcsL aggregate from pellet by treatment with unfolding buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 6 M guanidine hydrochloride for 30 min. For proper refolding, five times in volume refolding buffer, containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% glycerol, is added and left for overnight at 4°C on a stirrer rotor. Insoluble protein aggregates are removed by centrifugation and the supernatant containing soluble proteins is subjected to dialysis in dialysis buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% glycerol, to remove guanidine hydrochloride

Pseudomonas aeruginosa

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + H2O	-	Pseudomonas aeruginosa	ADP + phosphate	-	?
ATP + H2O	ATP binding structure with enzyme PscN, docking study	Pseudomonas aeruginosa	ADP + phosphate	-	?
ATP + H2O + cellular protein[side 1]	-	Pseudomonas aeruginosa	ADP + phosphate + cellular protein[side 2]	-	?
ATP + H2O + cellular protein[side 1]	-	Pseudomonas aeruginosa UCBPP-PA14	ADP + phosphate + cellular protein[side 2]	-	?
additional information	phosphate detection by malachite green assay	Pseudomonas aeruginosa	?	-	-

Subunits

Subunits	Comment	Organism
hexamer	PscN exists predominantly as oligomer	Pseudomonas aeruginosa
homohexamer	6 * 48000, SDS-PAGE	Pseudomonas aeruginosa
homohexamer	6 * 47900, calculated from amino acid sequence	Pseudomonas aeruginosa
More	the PscN primary structure shows three different domains: N-terminal (residues 1-95), central ATPase (residues 96-370), and C-terminal domain (residues 370-440), PscN tertiary structure analysis, overview	Pseudomonas aeruginosa

Synonyms

Synonyms	Comment	Organism
PscN	-	Pseudomonas aeruginosa
T3SS ATPase	-	Pseudomonas aeruginosa
type three secretion system ATPase	-	Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Pseudomonas aeruginosa

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
56.6	-	melting temperature	Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.8	-	assay at	Pseudomonas aeruginosa

General Information

General Information	Comment	Organism
additional information	PscN possesses G-X-X-P and G-X-P sequence motifs (X-any amino acid) in its primary sequences which are frequently found in the peptide channels and putative membrane ion channels. These motifs can be attributed to the N-terminal flexibility of the protein	Pseudomonas aeruginosa
physiological function	functional analysis of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa, PscL inhibits PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL)	Pseudomonas aeruginosa