Literature summary for 2.4.2.8 extracted from

Teran, D.; Hockova, D.; Cesnek, M.; Zikova, A.; Naesens, L.; Keough, D.T.; Guddat, L.W.
Crystal structures and inhibition of Trypanosoma brucei hypoxanthine-guanine phosphoribosyltransferase (2016), Sci. Rep., 6, 35894 .

Application

Application	Comment	Organism
drug development	structural differences between the Tbr and human enzymes suggest that selective inhibitors for the Tbr enzyme can be designed. Crystal structures of the enzyme in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors are determined. Differences occur between the diphosphate binding loops in human HGPRT and TbrHGPRT	Trypanosoma brucei brucei

Cloned(Commentary)

Cloned (Comment)	Organism
gene HGPRT, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3). The His6 tag does not appear to affect the activity of the enzyme	Trypanosoma brucei brucei

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant TbrHGPRT in complex with GMP, IMP, and inhibitors [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid, [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid, and [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid, hanging drop vapor diffusion method, mixing of 0.001 ml of protein ligand complex solution and 0.001 ml of well solution. The protein-ligand complexes are prepared using 36 mg/ml, 28 mg/ml, 60 mg/ml, and 28 mg/ml of enzyme in the presence of 3.2 mM GMP, 3.2 mM IMP, 4.6 mM [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid, 4.6 mM [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid and 1.8 mM [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid, respectively, followed by incubation on ice for 10 min, the reservoir solution for the complexes with GMP, IMP, [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid, and [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid contains 25% PEG 3350, 0.2 M lithium sulfate, and 0.1 M Bis-Tris, pH 5.0-5.5. For [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid the well solution is 25% PEG 3350, 0.2 M sodium iodide, 0.1 M Bis-Tris propane, pH 6.5, X-ray diffraction structure determination and analysis at 2.73, 2.51, 1.52, 2.89 and 2.81 A resolution, respectively. The structure of the complex with [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid is solved by molecular replacement using the structure of TcrHGPRT (PDB ID 1TC2) as the starting model. The starting models for the complexes with GMP, IMP, [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid, and [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid are based upon the refined model of the complex with [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid, refinement and model building	Trypanosoma brucei brucei

Crystallization (Comment)

Organism

purified recombinant TbrHGPRT in complex with GMP, IMP, and inhibitors [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid, [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid, and [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid, hanging drop vapor diffusion method, mixing of 0.001 ml of protein ligand complex solution and 0.001 ml of well solution. The protein-ligand complexes are prepared using 36 mg/ml, 28 mg/ml, 60 mg/ml, and 28 mg/ml of enzyme in the presence of 3.2 mM GMP, 3.2 mM IMP, 4.6 mM [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid, 4.6 mM [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid and 1.8 mM [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid, respectively, followed by incubation on ice for 10 min, the reservoir solution for the complexes with GMP, IMP, [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid, and [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid contains 25% PEG 3350, 0.2 M lithium sulfate, and 0.1 M Bis-Tris, pH 5.0-5.5. For [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid the well solution is 25% PEG 3350, 0.2 M sodium iodide, 0.1 M Bis-Tris propane, pH 6.5, X-ray diffraction structure determination and analysis at 2.73, 2.51, 1.52, 2.89 and 2.81 A resolution, respectively. The structure of the complex with [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid is solved by molecular replacement using the structure of TcrHGPRT (PDB ID 1TC2) as the starting model. The starting models for the complexes with GMP, IMP, [4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid, and [5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid are based upon the refined model of the complex with [6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid, refinement and model building

Trypanosoma brucei brucei

Inhibitors

Inhibitors	Comment	Organism
GMP	-	Trypanosoma brucei brucei
IMP	-	Trypanosoma brucei brucei
additional information	structural differences between the Tbr and human enzymes suggest that selective inhibitors for the Tbr enzyme can be designed. Crystal structures of the enzyme in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors are determined. Evaluation of acyclic nucleoside phosphonate inhibitors, overview	Trypanosoma brucei brucei
[3-(6-oxo-1,6-dihydro-9H-purin-9-yl)propyl]phosphonic acid	a competitive inhibitor	Trypanosoma brucei brucei
[4-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid	a competitive inhibitor	Trypanosoma brucei brucei
[4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl]phosphonic acid	a competitive inhibitor, enzyme binding structure analysis, crystal structure, overview. In the complex with the inhibitor there is no association with K145 located away from the purine base and the base itself rotated slightly compared to when GMP is bound, such that the distance between the 6-oxo group and this lysine is 3.9 A at its closest approach. A sulfate ion is also observed in the active site, and is located in the diphosphate binding pocket where it interacts with the amide nitrogen of G55, the side-chain of R179 and one magnesium ion. A second magnesium is located between E113 and D114, which is a Mg2+ binding site in the 6-oxopurine PRTs	Trypanosoma brucei brucei
[5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid	a competitive inhibitor	Trypanosoma brucei brucei
[5-(6-oxo-1,6-dihydro-9H-purin-9-yl)pentyl]phosphonic acid	a competitive inhibitor, enzyme binding structure analysis, crystal structure, overview	Trypanosoma brucei brucei
[6-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid	a competitive inhibitor, enzyme binding structure analysis, crystal structure, overview	Trypanosoma brucei brucei
[6-(6-oxo-1,6-dihydro-9H-purin-9-yl)hexyl]phosphonic acid	a competitive inhibitor	Trypanosoma brucei brucei

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.0023	-	guanine	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei
0.0055	-	hypoxanthine	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei
0.103	-	5-phospho-alpha-D-ribose 1-diphosphate	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Trypanosoma brucei brucei

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
48500	-	about, dimeric His6-tagged enzyme, sequence calculation	Trypanosoma brucei brucei
50400	-	recombinant His6-tagged enzyme dimer, gel filtration	Trypanosoma brucei brucei
106000	-	recombinant His6-tagged enzyme tetramer, gel filtration	Trypanosoma brucei brucei

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
guanine + 5-phospho-alpha-D-ribose 1-diphosphate	Trypanosoma brucei brucei	-	GMP + diphosphate	-	?
hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate	Trypanosoma brucei brucei	-	IMP + diphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Trypanosoma brucei brucei	Q07010	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis	Trypanosoma brucei brucei

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
guanine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Trypanosoma brucei brucei	GMP + diphosphate	-	?
hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate	-	Trypanosoma brucei brucei	IMP + diphosphate	-	?
additional information	xanthine is a poor substrate	Trypanosoma brucei brucei	?	-	-

Subunits

Subunits	Comment	Organism
dimer or tetramer	-	Trypanosoma brucei brucei
More	analysis of the quaternary structure of enzyme TbrHGPRT in solution, overview	Trypanosoma brucei brucei

Synonyms

Synonyms	Comment	Organism
6-oxopurine phosphoribosyltransferase	-	Trypanosoma brucei brucei
HGPRT	-	Trypanosoma brucei brucei
hypoxanthine-guanine phosphoribosyltransferase	-	Trypanosoma brucei brucei
PRT	-	Trypanosoma brucei brucei
Tb927.10.1400	locus name	Trypanosoma brucei brucei

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Trypanosoma brucei brucei

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
17.1	-	hypoxanthine	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei
23.8	-	guanine	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	assay at	Trypanosoma brucei brucei

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.0305	-	GMP	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei
0.0773	-	IMP	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei

General Information

General Information	Comment	Organism
metabolism	analysis of the molecular basis to understand the 6-oxopurine salvage in Trypansoma brucei	Trypanosoma brucei brucei
additional information	crystal structures of the enzyme in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors are determined. Structure comparisons, overview. One of the most important interactions between a 6-oxopurine PRT and the nucleoside monophosphate is the hydrogen bond between the 6-oxo group and a highly conserved lysine side-chain, K145 in TbrHGPRT. This bond is critical in discriminating the 6-oxopurine from a 6-aminopurine, such as adenine	Trypanosoma brucei brucei
physiological function	6-oxopurine phosphoribosyltransferase (PRT) is an enzyme central to the purine salvage pathway, whose activity is critical for the production of nucleotides GMP and IMP that are required for DNA/RNA synthesis within the protozoan parasite	Trypanosoma brucei brucei

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
3109	-	hypoxanthine	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei
14348	-	guanine	recombinant wild-type enzyme, pH 7.4, 25°C	Trypanosoma brucei brucei