Literature summary extracted from

Gao, X.; Zeng, J.; Yi, H.; Zhang, F.; Tang, B.; Tang, X.F.
Four inserts within the catalytic domain confer extra stability and activity to hyperthermostable pyrolysin from Pyrococcus furiosus (2017), Appl. Environ. Microbiol., 83, e03228-16 .

Cloned(Commentary)

EC Number	Cloned (Comment)	Organism
3.4.21.B55	expression in Escherichia coli BL21-CodonPlus	Pyrococcus furiosus

Protein Variants

EC Number	Protein Variants	Comment	Organism
3.4.21.B55	D132A	the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin	Pyrococcus furiosus
3.4.21.B55	D132A/D136A	the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme	Pyrococcus furiosus
3.4.21.B55	D132A/N134A	the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. In the presence of EGTA, the protein quantity of very unstable variants enzyme does not decrease as much as that of the wild-type enzyme following heat treatment at 95°C	Pyrococcus furiosus
3.4.21.B55	D132A/N134A/D136A	the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme	Pyrococcus furiosus
3.4.21.B55	D132N/D136N	the heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin	Pyrococcus furiosus
3.4.21.B55	D136A	the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin	Pyrococcus furiosus
3.4.21.B55	D138A/Q139A/E140A/D141A	the mutant enzyme displays heat resistance similar to that of the WT under nonchelating conditions but exhibits decreased residual activity compared to the wild-type enzyme following incubation with EGTA at 95°C. These results suggest that the residues downstream of motif 1 also contribute to calcium binding at the Ca3 site	Pyrococcus furiosus
3.4.21.B55	D161A	maturation defective variant	Pyrococcus furiosus
3.4.21.B55	D161A/D163A	the variant proform is capable of maturation, but the yield of mature enzyme is very low	Pyrococcus furiosus
3.4.21.B55	D161A/D163A/D165A	maturation defective variant	Pyrococcus furiosus
3.4.21.B55	D161A/D163A/D165A/S441A	the D161A/D163A/D165A/S441A proform degrades more than the the S441A proform at 95°C, confirming that the replacement of Asp161, Asp163, and Asp165 with Ala affects structural stability of the proform and makes it more susceptible to thermogenic hydrolysis	Pyrococcus furiosus
3.4.21.B55	D161A/D165A	maturation defective variant	Pyrococcus furiosus
3.4.21.B55	D161N/D163N/D165N	the variant proform is capable of maturation, but the yield of mature enzyme is very low. Heat inactivation profile at 95° is similar to wild-type enzyme, either in the absence or presence of EGTA. Similar activity levels as cpompared to wild-type enzyme	Pyrococcus furiosus
3.4.21.B55	D163A	the variant proform is capable of maturation, but the yield of mature enzyme is very low. Heat inactivation profile at 95° is similar to wild-type enzyme, either in the absence or presence of EGTA. Slightly lower activity levels as cpompared to wild-type enzyme	Pyrococcus furiosus
3.4.21.B55	D163A/D165A	the variant proform is capable of maturation, but the yield of mature enzyme is very low	Pyrococcus furiosus
3.4.21.B55	D165A	the variant proform is capable of maturation, but the yield of mature enzyme is very low	Pyrococcus furiosus
3.4.21.B55	D167A/D170A/E171A	maturation defective variant	Pyrococcus furiosus
3.4.21.B55	D818N/D820N	half-life at 95°C is 18 h compared to 12 h for wild-type enzyme	Pyrococcus furiosus
3.4.21.B55	F135R	the thermostability of the variant does not change under nonchelating conditions compared to that of the wild-type. The variant shows a lower level of residual activity than the wild-type enzyme following incubation with EGTA at 95°C. This result implies that a positively charged Arg residue in Dx[DN]xDG motif 1 affects calcium binding at the Ca3 site	Pyrococcus furiosus
3.4.21.B55	N134A	the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. The heat inactivation profile of the mutant enzyme is similar to that of the wild-type enzyme at 95°C. It retains lower residual activities than that of the wild-type enzyme following incubation with EGTA at 95°C. These data indicate that the mutated residue is involved in calcium binding at the Ca3 site, which is required to stabilize pyrolysin	Pyrococcus furiosus
3.4.21.B55	N134A/D136A	the purified mature form of the variant enzyme exhibits specific activity similar to that of wild-type enzyme. In the presence of EGTA, the protein quantity of very unstable variants enzyme does not decrease as much as that of the wild-type enzyme following heat treatment at 95°C	Pyrococcus furiosus
3.4.21.B55	R249E	the mutation within insert IS29 improves enzyme thermostability and resistance to salt-induced autocleavage. This is likely due to removing unfavorable electrostatic interactions involving Arg249. Half-life at 95°C is 18 h compared to 12 h for wild-type enzyme	Pyrococcus furiosus
3.4.21.B55	R249E/D818N/D820N	the mutant enzyme is both resistant to salt induced autocleavage and exhibits a remarkably long half-life of approximately 36 h at 95°C. Half-life at 95°C is 36 h compared to 12 h for wild-type enzyme	Pyrococcus furiosus

General Stability

EC Number	General Stability	Organism
3.4.21.B55	four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease	Pyrococcus furiosus

KM Value [mM]

EC Number	KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
3.4.21.B55	2.27	-	succinyl-AAPK-4-nitroanilide	90°C, pH 7.5	Pyrococcus furiosus

Organic Solvent Stability

EC Number	Organic Solvent	Comment	Organism
3.4.21.B55	SDS	mature insert-deletion variants are characterized and show that insert IS29 and insert IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites. One within insert IS29 and the other in insert IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation	Pyrococcus furiosus
3.4.21.B55	urea	mature insert-deletion variants are characterized and show that insert IS29 and insert IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites. One within insert IS29 and the other in insert IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation	Pyrococcus furiosus

Organism

EC Number	Organism	UniProt	Comment	Textmining
3.4.21.B55	Pyrococcus furiosus	-	-	-

Purification (Commentary)

EC Number	Purification (Comment)	Organism
3.4.21.B55	-	Pyrococcus furiosus

Substrates and Products (Substrate)

EC Number	Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
3.4.21.B55	succinyl-AAPK-4-nitroanilide + H2O	-	Pyrococcus furiosus	succinyl-AAPK + 4-nitroaniline	-	?

Temperature Stability [°C]

EC Number	Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
3.4.21.B55	additional information	-	extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments	Pyrococcus furiosus
3.4.21.B55	95	-	half-life: 12 h (wild-type enzyme), 18 h (mutant enzymes R249E and D818N/D820N), 36 h (R249E/D818N/D820N)	Pyrococcus furiosus

Turnover Number [1/s]

EC Number	Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
3.4.21.B55	1726	-	succinyl-AAPK-4-nitroanilide	90°C, pH 7.5	Pyrococcus furiosus