Literature summary for 2.5.1.15 extracted from

Chitnumsub, P.; Jaruwat, A.; Talawanich, Y.; Noytanom, K.; Liwnaree, B.; Poen, S.; Yuthavong, Y.
The structure of Plasmodium falciparum hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase reveals the basis of sulfa resistance (2020), FEBS J., 287, 3273-3297 .

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli Rosetta(DE3)pLysS cells	Plasmodium falciparum

Crystallization (Commentary)

Crystallization (Comment)	Organism
HPPK-DHPS from wild type and sulfa-resistant parasites, in complex with pteroate, sulfathiazole-dihydropteroate, and sulfadoxine-dihydropteroate, microbatch method, using 0.1 mM bicine pH 9.0, 0.5-0.6 M calcium acetate-Ca(OAc)2, 20% (w/v) PEG 4000 at 25°C	Plasmodium falciparum
structures of the enzyme from wild-type and sulfa-resistant mutants, both as apoenzyme and as complexes with pteroate and sulfa derivatives. Pteroate stays in active sites without steric constraint. In contrast, parts of the sulfa compounds situated outside of the substrate envelope are in the vicinity of the resistance mutations. Steric conflict between compound and mutant residue along with increased flexibility of loop D2 in the mutants can account for the reduced compound binding affinity to the mutants	Plasmodium falciparum

Crystallization (Comment)

Organism

HPPK-DHPS from wild type and sulfa-resistant parasites, in complex with pteroate, sulfathiazole-dihydropteroate, and sulfadoxine-dihydropteroate, microbatch method, using 0.1 mM bicine pH 9.0, 0.5-0.6 M calcium acetate-Ca(OAc)2, 20% (w/v) PEG 4000 at 25°C

Plasmodium falciparum

structures of the enzyme from wild-type and sulfa-resistant mutants, both as apoenzyme and as complexes with pteroate and sulfa derivatives. Pteroate stays in active sites without steric constraint. In contrast, parts of the sulfa compounds situated outside of the substrate envelope are in the vicinity of the resistance mutations. Steric conflict between compound and mutant residue along with increased flexibility of loop D2 in the mutants can account for the reduced compound binding affinity to the mutants

Plasmodium falciparum

Protein Variants

Protein Variants	Comment	Organism
A437G	mutant enzyme shows increased affinity for 4-aminobenzoate along with reduced binding and efficacy of sulfa drugs. (kcat/Km) for 4-aminobenzoate is increased by 6fold. Mutation enhances the dynamic flexibility of loop D2	Plasmodium falciparum
A437G/K540E	the mutations slightly decrease the catalytic efficiency (kcat/Km) of the enzyme with 4-aminobenzoate as substrate	Plasmodium falciparum
A581G	the mutation strongly decreases the catalytic efficiency (kcat/Km) of the enzyme with 4-aminobenzoate as substrate	Plasmodium falciparum
A613S	the mutation strongly decreases the catalytic efficiency (kcat/Km) of the enzyme with 4-aminobenzoate as substrate	Plasmodium falciparum
A613T	the mutation slightly decreases the catalytic efficiency (kcat/Km) of the enzyme with 4-aminobenzoate as substrate	Plasmodium falciparum
K540E	the mutation strongly decreases the catalytic efficiency (kcat/Km) of the enzyme with 4-aminobenzoate as substrate	Plasmodium falciparum
S436A/A437G	the mutations increase the catalytic efficiency (kcat/Km) of the enzyme by 1.6fold with 4-aminobenzoate as substrate	Plasmodium falciparum
S436F/A437G/A613S	the mutations increase the catalytic efficiency (kcat/Km) of the enzyme by 2fold with 4-aminobenzoate as substrate	Plasmodium falciparum
S436F/A437G/A613T	the triple mutant exhibits lower Km value for 4-aminobenzoate than the wild type enzyme. The mutations slightly increase the catalytic efficiency (kcat/Km) of the enzyme	Plasmodium falciparum
S436F/A613S	the mutations strongly decrease the catalytic efficiency (kcat/Km) of the enzyme with 4-aminobenzoate as substrate	Plasmodium falciparum
S436F/A613T	the mutations strongly decrease the catalytic efficiency (kcat/Km) of the enzyme with 4-aminobenzoate as substrate	Plasmodium falciparum

Inhibitors

Inhibitors	Comment	Organism
dapsone	inhibition of dihydropteroate synthase activity	Plasmodium falciparum
Pteroate	inhibition of dihydropteroate synthase activity	Plasmodium falciparum
sulfadimethoxine	inhibition of dihydropteroate synthase activity	Plasmodium falciparum
sulfadoxine	inhibition of dihydropteroate synthase activity	Plasmodium falciparum
sulfamethazine	inhibition of dihydropteroate synthase activity	Plasmodium falciparum
sulfamethoxazole	inhibition of dihydropteroate synthase activity	Plasmodium falciparum
sulfamonomethoxine	inhibition of dihydropteroate synthase activity	Plasmodium falciparum
sulfathiazole	inhibition of dihydropteroate synthase activity	Plasmodium falciparum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.00033	-	4-Aminobenzoate	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.00034	-	4-Aminobenzoate	mutant enzyme S436A/A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.00046	-	4-Aminobenzoate	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.00062	-	4-Aminobenzoate	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.00101	-	4-Aminobenzoate	mutant enzyme A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.00132	-	4-Aminobenzoate	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.00149	-	4-Aminobenzoate	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.00151	-	4-Aminobenzoate	mutant enzyme K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.00217	-	4-Aminobenzoate	mutant enzyme S436F/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.00223	-	4-Aminobenzoate	mutant enzyme A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.00245	-	4-Aminobenzoate	mutant enzyme S436F/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.00443	-	4-Aminobenzoate	mutant enzyme A581G, at pH 9.0 and 37°C	Plasmodium falciparum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	10 mM used in assay conditions	Plasmodium falciparum

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
86000	-	-	Plasmodium falciparum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
(7,8-dihydropterin-6-yl)methyl diphosphate + 4-aminobenzoate	Plasmodium falciparum	-	7,8-dihydropteroate + diphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Plasmodium falciparum	Q25704	bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase, cf. EC 2.7.6.3	-

Purification (Commentary)

Purification (Comment)	Organism
Ni-Sepharose column chromatography	Plasmodium falciparum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
(7,8-dihydropterin-6-yl)methyl diphosphate + 4-aminobenzoate	-	Plasmodium falciparum	7,8-dihydropteroate + diphosphate	-	?

Subunits

Subunits	Comment	Organism
?	x * 86000, SDS-PAGE	Plasmodium falciparum

Synonyms

Synonyms	Comment	Organism
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-dihydropteroate synthase	bifunctional enzyme	Plasmodium falciparum
7,8-dihydropteroate synthase	-	Plasmodium falciparum
HPPK-DHPS	bifunctional enzyme with hydroxymethyldihydropterin diphosphokinase and dihydropteroate synthase activities	Plasmodium falciparum
PPPK-DHPS	-	Plasmodium falciparum

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0057	-	4-Aminobenzoate	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.0075	-	4-Aminobenzoate	mutant enzyme S436A/A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0077	-	4-Aminobenzoate	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.01	-	4-Aminobenzoate	mutant enzyme A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.01	-	4-Aminobenzoate	mutant enzyme S436F/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.011	-	4-Aminobenzoate	mutant enzyme K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.012	-	4-Aminobenzoate	mutant enzyme A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.0137	-	4-Aminobenzoate	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.014	-	4-Aminobenzoate	mutant enzyme S436F/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.015	-	4-Aminobenzoate	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.02	-	4-Aminobenzoate	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.029	-	4-Aminobenzoate	mutant enzyme A581G, at pH 9.0 and 37°C	Plasmodium falciparum

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
0.0005	-	sulfathiazole	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.0006	-	Pteroate	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.0006	-	sulfamonomethoxine	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.0008	-	dapsone	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.0008	-	sulfamethazine	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.0008	-	sulfadimethoxine	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.0009	-	Pteroate	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.001	-	Pteroate	mutant enzyme S436A/A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0011	-	dapsone	mutant enzyme K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.0014	-	dapsone	mutant enzyme A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.0014	-	Pteroate	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.0016	-	sulfathiazole	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0016	-	sulfamethazine	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0017	-	Pteroate	mutant enzyme S436F/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.0018	-	sulfamonomethoxine	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0019	-	dapsone	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0024	-	sulfadimethoxine	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.003	-	Pteroate	mutant enzyme A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.003	-	Pteroate	mutant enzyme K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.0033	-	Pteroate	mutant enzyme A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.0037	-	Pteroate	mutant enzyme S436F/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.0038	-	Pteroate	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0039	-	dapsone	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.0052	-	sulfadoxine	mutant enzyme A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.0069	-	Pteroate	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.007	-	sulfadoxine	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.007	-	dapsone	mutant enzyme A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.0071	-	sulfamethazine	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.0071	-	sulfadoxine	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.0072	-	sulfathiazole	mutant enzyme S436A/A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.0091	-	sulfamonomethoxine	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.0092	-	sulfadoxine	mutant enzyme A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.014	-	sulfadimethoxine	mutant enzyme S436A/A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.014	-	sulfamonomethoxine	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.016	-	sulfadimethoxine	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.018	-	sulfathiazole	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.02	-	sulfadoxine	mutant enzyme S436A/A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.021	-	sulfamethazine	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.023	-	sulfadoxine	mutant enzyme K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.025	-	sulfathiazole	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.027	-	sulfathiazole	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.034	-	sulfamethoxazole	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
0.035	-	sulfamonomethoxine	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.039	-	Pteroate	mutant enzyme A581G, at pH 9.0 and 37°C	Plasmodium falciparum
0.04	-	dapsone	mutant enzyme A581G, at pH 9.0 and 37°C	Plasmodium falciparum
0.04	-	sulfadimethoxine	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.058	-	dapsone	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.058	-	sulfamethazine	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.07	-	sulfadoxine	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.084	-	sulfadoxine	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.086	-	sulfadoxine	mutant enzyme S436F/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.116	-	sulfadimethoxine	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.124	-	sulfadoxine	mutant enzyme S436F/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.153	-	sulfamethoxazole	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum
0.156	-	dapsone	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.177	-	dapsone	mutant enzyme S436F/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.192	-	dapsone	mutant enzyme S436F/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.196	-	sulfadoxine	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
0.198	-	sulfadoxine	mutant enzyme A581G, at pH 9.0 and 37°C	Plasmodium falciparum
0.462	-	sulfamethoxazole	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
0.48	-	sulfamethoxazole	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
0.731	-	sulfamethoxazole	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
4.08	-	4-Aminobenzoate	mutant enzyme S436F/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
5.38	-	4-Aminobenzoate	mutant enzyme A613S, at pH 9.0 and 37°C	Plasmodium falciparum
6.45	-	4-Aminobenzoate	mutant enzyme S436F/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
6.55	-	4-Aminobenzoate	mutant enzyme A581G, at pH 9.0 and 37°C	Plasmodium falciparum
7.28	-	4-Aminobenzoate	mutant enzyme K540E, at pH 9.0 and 37°C	Plasmodium falciparum
9.13	-	4-Aminobenzoate	mutant enzyme A437G/K540E, at pH 9.0 and 37°C	Plasmodium falciparum
9.9	-	4-Aminobenzoate	mutant enzyme A613T, at pH 9.0 and 37°C	Plasmodium falciparum
10.17	-	4-Aminobenzoate	wild type enzyme, at pH 9.0 and 37°C	Plasmodium falciparum
10.35	-	4-Aminobenzoate	mutant enzyme S436F/A437G/A613T, at pH 9.0 and 37°C	Plasmodium falciparum
16.67	-	4-Aminobenzoate	mutant enzyme S436F/A437G/A613S, at pH 9.0 and 37°C	Plasmodium falciparum
21.43	-	4-Aminobenzoate	mutant enzyme S436A/A437G, at pH 9.0 and 37°C	Plasmodium falciparum
60.6	-	4-Aminobenzoate	mutant enzyme A437G, at pH 9.0 and 37°C	Plasmodium falciparum