Literature summary for 2.7.7.84 extracted from

Hartmann, R.; Justesen, J.; Sarkar, S.N.; Sen, G.C.; Yee, V.C.
Crystal structure of the 2'-specific and double-stranded RNA-activated interferon-induced antiviral protein 2'-5'-oligoadenylate synthetase (2003), Mol. Cell, 12, 1173-1185.

Activating Compound

Activating Compound	Comment	Organism	Structure
dsRNA	2'-5'-oligoadenylate synthases are activated by viral double-stranded RNA in infected cells and initiate a cellular response by synthesizing 2'-5'-oligoadenylates, which in turn activate RNase L. All mammalian OAS proteins require dsRNA for activity ssRNA or DNA does not activate this class of enzymes. The dsRNA activator must be at least 15 nucleotides long, and no modification of the 2'-hydroxyl group is tolerated. OAS1 RNA activation site structure, overview	Sus scrofa
viral dsRNA	2'-5'-oligoadenylate synthases are activated by viral double-stranded RNA in infected cells and initiate a cellular response by synthesizing 2'-5'-oligoadenylates, which in turn activate RNase L. All mammalian OAS proteins require dsRNA for activity ssRNA or DNA does not activate this class of enzymes. The dsRNA activator must be at least 15 nucleotides long, and no modification of the 2'-hydroxyl group is tolerated. OAS1 RNA activation site structure, overview	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
OAS1, expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Sus scrofa

Crystallization (Commentary)

Crystallization (Comment)	Organism
OAS1, purified recombinant porcine OAS1, 1.5 mg/ml protein in 50 mM NaCl, 10 mM HEPES, pH 6.8, 0.5 mM EDTA, with 5 mM of iodoacetamide for 30 min at room temperature, then concentrated to 6 mg/ml for crystallization by vapor diffusion, best crystals grow at 20°C in 1:1 drops with a well solution of 30% PEG 2000 MME, 0.2 M ammonium sulfate, and 0.1 M sodium cacodylate at pH 6, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement	Sus scrofa
OAS1, X-ray diffraction structure determination and analysis	Homo sapiens

Crystallization (Comment)

Organism

OAS1, purified recombinant porcine OAS1, 1.5 mg/ml protein in 50 mM NaCl, 10 mM HEPES, pH 6.8, 0.5 mM EDTA, with 5 mM of iodoacetamide for 30 min at room temperature, then concentrated to 6 mg/ml for crystallization by vapor diffusion, best crystals grow at 20°C in 1:1 drops with a well solution of 30% PEG 2000 MME, 0.2 M ammonium sulfate, and 0.1 M sodium cacodylate at pH 6, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement

Sus scrofa

OAS1, X-ray diffraction structure determination and analysis

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
K203E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
K212A	the mutant has strongly impaired catalytic activity, KM for ATP is increased by almost 4fold relative to that of the wild-type protein, and kcat is decreased by roughly 8fold	Sus scrofa
K41E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
K59E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R194E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R194E/R198E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R198E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R198M	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R245E/K246E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R38E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R38E/K41E	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Sus scrofa
R38E/K41E/K59E/R194E/R198E	site-directed mutagenesis, the mutant is almost inactive	Sus scrofa
S62A	the mutant displays a strong substrate inhibition at higher ATP concentrations, more dramatic than that observed for wild-type OAS1	Sus scrofa
S63A	the mutant displays a strong substrate inhibition at higher ATP concentrations, more dramatic than that observed for wild-type OAS1	Sus scrofa

Inhibitors

Inhibitors	Comment	Organism	Structure
ATP	substrate inhibition at higher concentrations	Sus scrofa

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.00044	-	ATP	wild-type OAS1, with ATP as sole substrate, pH and temperature not specified in the publication	Sus scrofa
0.0011	-	NAD+	OAS1 mutant S62A, with ATP and NAD+ as substrates, pH and temperature not specified in the publication	Sus scrofa
0.0013	-	NAD+	wild-type OAS1, with ATP and NAD+ as substrates, pH and temperature not specified in the publication	Sus scrofa
0.0014	-	NAD+	OAS1 mutant S63A, with ATP and NAD+ as substrates, pH and temperature not specified in the publication	Sus scrofa
0.0017	-	ATP	OAS1 mutant K212A, with ATP as sole substrate, pH and temperature not specified in the publication	Sus scrofa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
3 ATP	Homo sapiens	-	pppA2'p5'A2'p5'A + 2 diphosphate	-	?
3 ATP	Sus scrofa	-	pppA2'p5'A2'p5'A + 2 diphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	isozyme OAS1	-
Sus scrofa	-	-	-
Sus scrofa	Q29599	isozyme OAS1	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant OAS1 from Escherichia coli strain BL21(DE3) by gel filtration, ammonium sulfate fractionation, heparin affinity chromatography and gel filtration	Sus scrofa

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.0005	-	OAS1 mutant R38E/K41E/K59E/R194E/R198E, pH and temperature not specified in the publication	Sus scrofa
0.001	-	OAS1 mutant R194E/R198E, pH and temperature not specified in the publication	Sus scrofa
0.004	-	OAS1 mutant R198E, pH and temperature not specified in the publication	Sus scrofa
0.007	-	OAS1 mutant R198M, pH and temperature not specified in the publication	Sus scrofa
0.016	-	OAS1 mutant R38E/K41E, pH and temperature not specified in the publication	Sus scrofa
0.05	-	OAS1 mutant K203E, pH and temperature not specified in the publication	Sus scrofa
0.117	-	OAS1 mutant K59E, pH and temperature not specified in the publication	Sus scrofa
0.16	-	OAS1 mutant K41E, pH and temperature not specified in the publication	Sus scrofa
0.17	-	OAS1 mutant R194E, pH and temperature not specified in the publication	Sus scrofa
0.22	-	OAS1 mutant R38E, pH and temperature not specified in the publication	Sus scrofa
3.3	-	OAS1 mutant R245E/K246E, pH and temperature not specified in the publication	Sus scrofa
10.1	-	wild-type OAS1, pH and temperature not specified in the publication	Sus scrofa

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3 ATP	-	Homo sapiens	pppA2'p5'A2'p5'A + 2 diphosphate	-	?
3 ATP	-	Sus scrofa	pppA2'p5'A2'p5'A + 2 diphosphate	-	?
dATP + NAD+	-	Sus scrofa	?	-	?

Synonyms

Synonyms	Comment	Organism
2'-5'-oligoadenylate synthetase	-	Homo sapiens
2'-5'-oligoadenylate synthetase	-	Sus scrofa
OAS	-	Homo sapiens
OAS	-	Sus scrofa

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.95	-	ATP	OAS1 mutant K212A, with ATP as sole substrate, pH and temperature not specified in the publication	Sus scrofa
0.99	-	NAD+	OAS1 mutant S62A, with ATP and NAD+ as substrates, pH and temperature not specified in the publication	Sus scrofa
3.6	-	NAD+	OAS1 mutant S63A, with ATP and NAD+ as substrates, pH and temperature not specified in the publication	Sus scrofa
3.6	-	NAD+	wild-type OAS1, with ATP and NAD+ as substrates, pH and temperature not specified in the publication	Sus scrofa
8.1	-	ATP	wild-type OAS1, with ATP as sole substrate, pH and temperature not specified in the publication	Sus scrofa

Expression

Organism	Comment	Expression
Homo sapiens	induction by interferon	up
Sus scrofa	induction by interferon	up

General Information

General Information	Comment	Organism
evolution	conserved catalytic mechanism for the 2'- and 3'-specific nucleotidyl transferases. specific nucleotidyl transferases. Comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions	Homo sapiens
evolution	conserved catalytic mechanism for the 2'- and 3'-specific nucleotidyl transferases. specific nucleotidyl transferases. Comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions	Sus scrofa
malfunction	mutation of the conserved Leu3 and Pro7 and of Cys330, Cys331, and Lys332 reduce enzyme activity	Homo sapiens
malfunction	mutation of the conserved Leu3 and Pro7 and of Cys330, Cys331, and Lys332 reduce enzyme activity. Mutants S62A and S63A display Michaelis-Menten kinetics toward NAD+, the kcat of the Ser62Ala mutant is approximately 3fold lower than the kcat for either the wild-type or the S63A mutant	Sus scrofa
additional information	OAS active site structure with three conserved active site aspartic acid residues, overview	Homo sapiens
additional information	OAS active site structure with three conserved active site aspartic acid residues, overview	Sus scrofa
physiological function	2'-5'-oligoadenylate synthases are interferon-induced, double-stranded RNA-activated antiviral enzymes which are the only proteins known to catalyze 2'-specific nucleotidyl transfer	Homo sapiens
physiological function	2'-5'-oligoadenylate synthases are interferon-induced, double-stranded RNA-activated antiviral enzymes which are the only proteins known to catalyze 2'-specific nucleotidyl transfer	Sus scrofa