Literature summary for 2.4.2.7 extracted from

Arco, J.D.; Perez, E.; Naitow, H.; Matsuura, Y.; Kunishima, N.; Fernandez-Lucas, J.
Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5-monophospate analogues (2019), Biores. Technol., 276, 244-252 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene ttha1614 or aprt2, recombinant overexpression of His6-tagged TtAPRT in Escherichia coli strain BL21(DE3) or recombinant expression of nontagged enzyme in Escherichia coli strain BL21 Codon Plus (DE3)-RIL	Thermus thermophilus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme APRT2, hanging-drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 200 mM NaCl and 20 mM Tris-HCl, pH 8.0, with 0.001 ml of precipitant solution containing 19% PEG 20000 and 0.1 M sodium citrate, pH 6.0, 30-50 days, 20°C, X-ray diffraction structure determination and analysis at 2.6 A resolution, molecular replacement using the structure of TthAPRT1 from Thermus thermophilus HB8 (PDB ID 1VCH) as a template	Thermus thermophilus

Crystallization (Comment)

Organism

purified recombinant enzyme APRT2, hanging-drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 200 mM NaCl and 20 mM Tris-HCl, pH 8.0, with 0.001 ml of precipitant solution containing 19% PEG 20000 and 0.1 M sodium citrate, pH 6.0, 30-50 days, 20°C, X-ray diffraction structure determination and analysis at 2.6 A resolution, molecular replacement using the structure of TthAPRT1 from Thermus thermophilus HB8 (PDB ID 1VCH) as a template

Thermus thermophilus

Protein Variants

Protein Variants	Comment	Organism
additional information	covalent immobilization of TtAPRT2 through surface exposed Lys residues promotes a multipoint covalent attachment which leads to higher degree of rigidification, thereby increasing the thermal stability of the protein. Dimeric TtAPRT2 is immobilized onto glutaraldehyde-activated magnetic iron oxide porous microparticles by two different strategies: (a) an enzyme immobilization at pH 8.5 to encourage the immobilization process by N-termini (MTtAPRT2A, MTtAPRT2B, MTtAPRT2C) or (b) an enzyme immobilization at pH 10.0 to encourage the immobilization process through surface exposed lysine residues (MTtAPRT2D, MTtAPRT2E, MTtAPRT2F). According to catalyst load experiments, MTtAPRT2B (activity: 480 IU/g biocatalyst, activity recovery 52%) and MTtAPRT2F (activity 507 IU/g biocatalyst, activity recovery 44%) are chosen as optimal derivatives. The potential reusability of MTtAPRT2B and MTtAPRT2F is also tested. Finally, MTtAPRT2F is employed in the synthesis of nucleoside-5'-monophosphate analogues. 0.025 ml of the bead suspension (0.020 mg/ml) are washed and equilibrated in corresponding binding buffer containing 50 mM potassium phosphate buffer, pH 8.5, 50 mM sodium borate buffer, pH 10.0, or 50 mM sodium borate buffer, pH 10.6, during 4 h at 25°C	Thermus thermophilus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Thermus thermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	Thermus thermophilus	-	AMP + diphosphate	-	?
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	Thermus thermophilus DSM 579	-	AMP + diphosphate	-	?
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	Thermus thermophilus ATCC 27634	-	AMP + diphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Thermus thermophilus	Q5SHW6	chains A-F	-
Thermus thermophilus ATCC 27634	Q5SHW6	chains A-F	-
Thermus thermophilus DSM 579	Q5SHW6	chains A-F	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged TtAPRT from Escherichia coli strain BL21(DE3) by nickel affinity chromatograph, tag cleavage by thrombin, and gel filtration. Recombinant nontagged enzyme from Escherichia coli strain BL21 Codon Plus (DE3)-RIL by heat treatment at 70°C for 15 min in a digital sonifier, two different steps of anion exchange chromatography, intervening desalting gel filtration, and hydroxyapatite chromatography, followed by ultrafiltration, and gel filtration	Thermus thermophilus

Purification (Comment)

Organism

recombinant His6-tagged TtAPRT from Escherichia coli strain BL21(DE3) by nickel affinity chromatograph, tag cleavage by thrombin, and gel filtration. Recombinant nontagged enzyme from Escherichia coli strain BL21 Codon Plus (DE3)-RIL by heat treatment at 70°C for 15 min in a digital sonifier, two different steps of anion exchange chromatography, intervening desalting gel filtration, and hydroxyapatite chromatography, followed by ultrafiltration, and gel filtration

Thermus thermophilus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
90	-	substrate 6-methylpurine, pH 8.0, 70°C, recombinant enzyme	Thermus thermophilus
103	-	substrate 6-methoxyguanine, pH 8.0, 70°C, recombinant enzyme	Thermus thermophilus
405	-	substrate 2-chloroadenine, pH 8.0, 70°C, recombinant enzyme	Thermus thermophilus
641	-	substrate 2,6-diaminopurine, pH 8.0, 70°C, recombinant enzyme	Thermus thermophilus
785	-	substrate 2-fluoroadenine, pH 8.0, 70°C, recombinant enzyme	Thermus thermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,6-diaminopurine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus	2,6-diaminopurine nucleoside-5'-monophosphate + diphosphate	-	r
2,6-diaminopurine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus DSM 579	2,6-diaminopurine nucleoside-5'-monophosphate + diphosphate	-	r
2,6-diaminopurine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus ATCC 27634	2,6-diaminopurine nucleoside-5'-monophosphate + diphosphate	-	r
2-chloroadenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus	2-chloro-AMP + diphosphate	-	r
2-chloroadenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus DSM 579	2-chloro-AMP + diphosphate	-	r
2-chloroadenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus ATCC 27634	2-chloro-AMP + diphosphate	-	r
2-fluoroadenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus	2-fluoro-AMP + diphosphate	-	r
6-methoxyguanine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus	6-methoxyguanosine 5'-monophosphate + diphosphate	-	r
6-methylpurine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus	6-methylpurine ribonucleoside-5'-monophosphate + diphosphate	-	r
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus	AMP + diphosphate	-	?
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus	AMP + diphosphate	-	r
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus DSM 579	AMP + diphosphate	-	?
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus DSM 579	AMP + diphosphate	-	r
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus ATCC 27634	AMP + diphosphate	-	?
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Thermus thermophilus ATCC 27634	AMP + diphosphate	-	r
additional information	no activity with 6-oxopurines (7-deazaxanthine and 7-deaza-6-hydroxypurine), 6-thiopurines (6-mercaptopurine), and 6-halopurines (6-chloropurine)	Thermus thermophilus	?	-	-
additional information	no activity with 6-oxopurines (7-deazaxanthine and 7-deaza-6-hydroxypurine), 6-thiopurines (6-mercaptopurine), and 6-halopurines (6-chloropurine)	Thermus thermophilus DSM 579	?	-	-
additional information	no activity with 6-oxopurines (7-deazaxanthine and 7-deaza-6-hydroxypurine), 6-thiopurines (6-mercaptopurine), and 6-halopurines (6-chloropurine)	Thermus thermophilus ATCC 27634	?	-	-

Subunits

Subunits	Comment	Organism
homodimer	TtAPRT2 structure analysis and comparisons. The asymmetric unit of TtAPRT2 crystal (PDB ID 5ZGO) contains six subunit chains A-F that comprise three identical homodimers AB, CD, and EF, all residues from N- to C-termini are modeled completely in all the six subunits of 5ZGO. Active site architecture shows that TtAPRT2 is formed by three domains that are common in all type APRTs, overview	Thermus thermophilus

Synonyms

Synonyms	Comment	Organism
adenine phosphoribosyltransferase 2	-	Thermus thermophilus
APRT2	-	Thermus thermophilus
TtAPRT2	-	Thermus thermophilus
TTHA1614	-	Thermus thermophilus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
75	-	recombinant enzyme	Thermus thermophilus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	the immobilization process improved the thermostability of TtAPRT2	Thermus thermophilus
60	-	purified enzyme, 15 h, pH 8.0, loss of 10% activity	Thermus thermophilus
70	-	purified enzyme, 15 h, pH 8.0, loss of 30% activity	Thermus thermophilus
80	-	purified enzyme, 15 h, pH 8.0, loss of 95% activity	Thermus thermophilus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.5	-	recombinant enzyme	Thermus thermophilus

General Information

General Information	Comment	Organism
physiological function	adenine phosphoribosyltransferase, APRT catalyzes the transfer of the 5-phosphoribosyl group from 5-phospho-alpha-D-ribosyl-1-diophosphate (PRPP) to N9 in 6-aminopurines, such as adenine or 6-aminopurine derivatives (e.g. 2,6-diaminopurine, 6-methylpurine, 2-fluoroadenine, among others), in presence of Mg2+ to obtain the corresponding NMPs	Thermus thermophilus