Literature summary for 2.4.2.7 extracted from

Pavithra, G.C.; Ramagopal, U.A.
Crystal structures of APRT from Francisella tularensis - an N-H-N hydrogen bond imparts adenine specificity in adenine phosphoribosyltransferases (2018), FEBS J., 285, 2306-2318 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene apt, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Francisella tularensis subsp. tularensis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme in apoform and in complex with substrate adenine, sitting drop vapor diffusion method, mixing of 10 mg/ml protein in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% glycerol, with crystallization solutions (a) 0.1 M Tris/HCl pH 8.5, 30% PEG 4000 and 0.2 M MgCl2 and (b) 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl, pH 8.5, 30% PEG 4000, X-ray diffraction structure determination and analysis at 1.9-2.28 A resolution	Francisella tularensis subsp. tularensis

Crystallization (Comment)

Organism

purified recombinant enzyme in apoform and in complex with substrate adenine, sitting drop vapor diffusion method, mixing of 10 mg/ml protein in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% glycerol, with crystallization solutions (a) 0.1 M Tris/HCl pH 8.5, 30% PEG 4000 and 0.2 M MgCl2 and (b) 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl, pH 8.5, 30% PEG 4000, X-ray diffraction structure determination and analysis at 1.9-2.28 A resolution

Francisella tularensis subsp. tularensis

Protein Variants

Protein Variants	Comment	Organism
F23A	site-directed mutagenesis, the mutation on the base-binding loop severely affects the activity and efficiency of the enzyme, the mutant enzyme is about 200fold less active as compared with wild-type	Francisella tularensis subsp. tularensis

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Francisella tularensis subsp. tularensis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	Francisella tularensis subsp. tularensis	-	AMP + diphosphate	-	r
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	Francisella tularensis subsp. tularensis Schu 4	-	AMP + diphosphate	-	r

Organism

Organism	UniProt	Comment	Textmining
Francisella tularensis subsp. tularensis	Q5NII9	-	-
Francisella tularensis subsp. tularensis Schu 4	Q5NII9	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage through thrombin, followed by dialysis	Francisella tularensis subsp. tularensis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.0056	-	recombinant mutant F23A enzyme, pH 7.5, 30-34°C	Francisella tularensis subsp. tularensis
1.15	-	recombinant wild-type enzyme, pH 7.5, 30-34°C	Francisella tularensis subsp. tularensis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Francisella tularensis subsp. tularensis	AMP + diphosphate	-	r
adenine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Francisella tularensis subsp. tularensis Schu 4	AMP + diphosphate	-	r
additional information	APRT differentiates adenine from other purines	Francisella tularensis subsp. tularensis	?	-	-
additional information	APRT differentiates adenine from other purines	Francisella tularensis subsp. tularensis Schu 4	?	-	-

Subunits

Subunits	Comment	Organism
homodimer	-	Francisella tularensis subsp. tularensis

Synonyms

Synonyms	Comment	Organism
APRT	-	Francisella tularensis subsp. tularensis
apT	-	Francisella tularensis subsp. tularensis
FtAPRT	-	Francisella tularensis subsp. tularensis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	34	assay at	Francisella tularensis subsp. tularensis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Francisella tularensis subsp. tularensis

General Information

General Information	Comment	Organism
metabolism	APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides	Francisella tularensis subsp. tularensis
additional information	the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H-N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. The residues conferring rigidity to the base-binding loop are highly conserved. Comparison of structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. The base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine. FtAPRT structure is divided into (a) the base-binding domain, (b) the core PRPP binding domain and (c) a flexible catalytic loop, which is proposed to sequester the active site from the solvent at the time of catalysis. Enzyme residue F23 is a key residue that stacks with the F16-P17 cis-peptide pair and stabilizes the base-binding loop. This residue also plays an important role by stacking against the substrate adenine	Francisella tularensis subsp. tularensis
physiological function	APRT is the sole enzyme with the crucial role of recycling (or salvaging) freely available adenine into AMP and exists in all phyla of life	Francisella tularensis subsp. tularensis