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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.2.7 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)

EC Number Crystallization (Comment) Organism
2.4.2.7 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

EC Number Protein Variants Comment Organism
2.4.2.7 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

EC Number Metals/Ions Comment Organism Structure
2.4.2.7 Mg2+ required Thermus thermophilus

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.2.7 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]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.4.2.7 90
-
 substrate 6-methylpurine, pH 8.0, 70°C, recombinant enzyme Thermus thermophilus
2.4.2.7 103
-
 substrate 6-methoxyguanine, pH 8.0, 70°C, recombinant enzyme Thermus thermophilus
2.4.2.7 405
-
 substrate 2-chloroadenine, pH 8.0, 70°C, recombinant enzyme Thermus thermophilus
2.4.2.7 641
-
 substrate 2,6-diaminopurine, pH 8.0, 70°C, recombinant enzyme Thermus thermophilus
2.4.2.7 785
-
 substrate 2-fluoroadenine, pH 8.0, 70°C, recombinant enzyme Thermus thermophilus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.2.7 2,6-diaminopurine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus 2,6-diaminopurine nucleoside-5'-monophosphate + diphosphate
-
 r
2.4.2.7 2,6-diaminopurine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus DSM 579 2,6-diaminopurine nucleoside-5'-monophosphate + diphosphate
-
 r
2.4.2.7 2,6-diaminopurine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus ATCC 27634 2,6-diaminopurine nucleoside-5'-monophosphate + diphosphate
-
 r
2.4.2.7 2-chloroadenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus 2-chloro-AMP + diphosphate
-
 r
2.4.2.7 2-chloroadenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus DSM 579 2-chloro-AMP + diphosphate
-
 r
2.4.2.7 2-chloroadenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus ATCC 27634 2-chloro-AMP + diphosphate
-
 r
2.4.2.7 2-fluoroadenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus 2-fluoro-AMP + diphosphate
-
 r
2.4.2.7 6-methoxyguanine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus 6-methoxyguanosine 5'-monophosphate + diphosphate
-
 r
2.4.2.7 6-methylpurine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus 6-methylpurine ribonucleoside-5'-monophosphate + diphosphate
-
 r
2.4.2.7 adenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus AMP + diphosphate
-
 ?
2.4.2.7 adenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus AMP + diphosphate
-
 r
2.4.2.7 adenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus DSM 579 AMP + diphosphate
-
 ?
2.4.2.7 adenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus DSM 579 AMP + diphosphate
-
 r
2.4.2.7 adenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus ATCC 27634 AMP + diphosphate
-
 ?
2.4.2.7 adenine + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Thermus thermophilus ATCC 27634 AMP + diphosphate
-
 r
2.4.2.7 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 ?
-
-
2.4.2.7 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 ?
-
-
2.4.2.7 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

EC Number Subunits Comment Organism
2.4.2.7 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

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.2.7 75
-
 recombinant enzyme Thermus thermophilus

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.4.2.7 additional information
-
 the immobilization process improved the thermostability of TtAPRT2 Thermus thermophilus
2.4.2.7 60
-
 purified enzyme, 15 h, pH 8.0, loss of 10% activity Thermus thermophilus
2.4.2.7 70
-
 purified enzyme, 15 h, pH 8.0, loss of 30% activity Thermus thermophilus
2.4.2.7 80
-
 purified enzyme, 15 h, pH 8.0, loss of 95% activity Thermus thermophilus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.2.7 8.5
-
 recombinant enzyme Thermus thermophilus

General Information

EC Number General Information Comment Organism
2.4.2.7 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