Literature summary for 1.5.1.33 extracted from

Labine, M.; DePledge, L.; Feirer, N.; Greenwich, J.; Fuqua, C.; Allen, K.
Enzymatic and mutational analysis of the PruA pteridine reductase required for pterin-dependent control of biofilm formation in Agrobacterium tumefaciens (2020), J. Bacteriol., 202, e00098-20 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene Atu1130, recombinant overexpression of N-terminally His6-tagged enzyme in Escherichia coli	Agrobacterium fabrum
gene ptr1, recombinant overexpression of N-terminally His6-tagged enzyme in Escherichia coli	Leishmania major

Protein Variants

Protein Variants	Comment	Organism
additional information	mutational analysis of the PruA YX3K motif. Generation of an Agrobacterium. tumefaciens DELTApruA strain	Agrobacterium fabrum
R24A	site-directed mutagenesis, reduced activity compared to wild-type	Agrobacterium fabrum
R24G	site-directed mutagenesis, highly reduced activity compared to wild-type	Agrobacterium fabrum
Y161A	site-directed mutagenesis, slightly reduced activity compared to wild-type	Agrobacterium fabrum
Y161A/Y163F	site-directed mutagenesis, inactive mutant	Agrobacterium fabrum
Y161F/Y163F	site-directed mutagenesis, reduced activity compared to wild-type	Agrobacterium fabrum
Y163A	site-directed mutagenesis, inactive mutant	Agrobacterium fabrum
Y163F	site-directed mutagenesis, slightly reduced catalytic efficiency but increased activity compared to wild-type	Agrobacterium fabrum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.0347	-	7,8-dihydrobiopterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum
0.0713	-	7,8-dihydromonapterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum
0.0951	-	7,8-dihydroneopterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum
0.1694	-	7,8-dihydromonapterin	recombinant mutant R24A, pH 6.0, 25°C	Agrobacterium fabrum
0.2127	-	7,8-dihydromonapterin	recombinant mutant Y163F, pH 6.0, 25°C	Agrobacterium fabrum
0.3332	-	7,8-dihydromonapterin	recombinant mutant Y161F/Y163F, pH 6.0, 25°C	Agrobacterium fabrum
0.505	-	7,8-dihydromonapterin	recombinant mutant Y161A, pH 6.0, 25°C	Agrobacterium fabrum

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
60000	-	gel filtration	Agrobacterium fabrum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
7,8-dihydrobiopterin + NADPH + H+	Leishmania major	-	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	Agrobacterium fabrum	-	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	Agrobacterium fabrum C58	-	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	Agrobacterium fabrum ATCC 33970	-	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
biopterin + NADPH + H+	Leishmania major	-	7,8-dihydrobiopterin + NADP+	-	?

Organism

Organism	UniProt	Comment	Textmining
Agrobacterium fabrum	Q7CZX3	Agrobacterium tumefaciens strain C58	-
Agrobacterium fabrum ATCC 33970	Q7CZX3	Agrobacterium tumefaciens strain C58	-
Agrobacterium fabrum C58	Q7CZX3	Agrobacterium tumefaciens strain C58	-
Leishmania major	Q01782	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme from Escherichia coli by nickel affinity chromatography, and gel filtration	Leishmania major
recombinant His6-tagged enzyme from Escherichia coli by nickel affinity chromatography, and gel filtration	Agrobacterium fabrum

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.28	-	recombinant mutant R24G, pH 6.0, 25°C, substrate 7,8-dihydromonapterin	Agrobacterium fabrum
1.71	-	recombinant mutant R24A, pH 6.0, 25°C, substrate 7,8-dihydromonapterin	Agrobacterium fabrum
2.88	-	recombinant mutant Y161A, pH 6.0, 25°C, substrate 7,8-dihydromonapterin	Agrobacterium fabrum
4.13	-	recombinant mutant Y161F/Y163F, pH 6.0, 25°C, substrate 7,8-dihydromonapterin	Agrobacterium fabrum
4.77	-	recombinant wild-type enzyme, pH 6.0, 25°C, substrate 7,8-dihydromonapterin	Agrobacterium fabrum
5.03	-	recombinant wild-type enzyme, pH 6.0, 25°C, substrate 7,8-dihydroneopterin	Agrobacterium fabrum
8.84	-	recombinant mutant Y163F, pH 6.0, 25°C, substrate 7,8-dihydromonapterin	Agrobacterium fabrum
9.83	-	recombinant wild-type enzyme, pH 6.0, 25°C, substrate 7,8-dihydrobiopterin	Agrobacterium fabrum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
7,8-dihydrobiopterin + NADPH + H+	-	Leishmania major	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	-	Agrobacterium fabrum	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	best substrate	Agrobacterium fabrum	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	low activity	Leishmania major	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	-	Agrobacterium fabrum C58	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	best substrate	Agrobacterium fabrum C58	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	-	Agrobacterium fabrum ATCC 33970	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrobiopterin + NADPH + H+	best substrate	Agrobacterium fabrum ATCC 33970	5,6,7,8-tetrahydrobiopterin + NADP+	-	?
7,8-dihydrofolate + NADPH + H+	low activity	Leishmania major	5,6,7,8-tetrahydrofolate + NADP+	-	?
7,8-dihydromonapterin + NADPH + H+	cf. EC 1.5.1.50, moderate activity	Agrobacterium fabrum	5,6,7,8-tetrahydromonapterin + NADP+	-	?
7,8-dihydromonapterin + NADPH + H+	cf. EC 1.5.1.50, moderate activity	Agrobacterium fabrum C58	5,6,7,8-tetrahydromonapterin + NADP+	-	?
7,8-dihydromonapterin + NADPH + H+	cf. EC 1.5.1.50, moderate activity	Agrobacterium fabrum ATCC 33970	5,6,7,8-tetrahydromonapterin + NADP+	-	?
7,8-dihydroneopterin + NADPH + H+	stereoisomer of biopterin, moderate activity	Agrobacterium fabrum	5,6,7,8-tetrahydroneopterin + NADP+	-	?
7,8-dihydroneopterin + NADPH + H+	stereoisomer of biopterin, moderate activity	Agrobacterium fabrum C58	5,6,7,8-tetrahydroneopterin + NADP+	-	?
7,8-dihydroneopterin + NADPH + H+	stereoisomer of biopterin, moderate activity	Agrobacterium fabrum ATCC 33970	5,6,7,8-tetrahydroneopterin + NADP+	-	?
biopterin + NADPH + H+	-	Leishmania major	7,8-dihydrobiopterin + NADP+	-	?
biopterin + NADPH + H+	best substrate	Leishmania major	7,8-dihydrobiopterin + NADP+	-	?
folate + NADPH + H+	high activity, see also EC 1.5.1.3	Leishmania major	7,8-dihydrofolate + NADP+	-	?
additional information	substrate specificity, overview	Leishmania major	?	-	-
additional information	enzyme PruA does not exhibit pteridine reductase activity with 7,8-dihydrofolate or fully oxidized pterins. PruA exhibits maximal catalytic efficiency with H2BPt, while both H2MPt and 7,8-dihydroneopterin (H2NPt) also serve as competent substrates. Substrate specificity, overview. No activity with 7,8-dihydrofolate, biopterin, and folate	Agrobacterium fabrum	?	-	-
additional information	enzyme PruA does not exhibit pteridine reductase activity with 7,8-dihydrofolate or fully oxidized pterins. PruA exhibits maximal catalytic efficiency with H2BPt, while both H2MPt and 7,8-dihydroneopterin (H2NPt) also serve as competent substrates. Substrate specificity, overview. No activity with 7,8-dihydrofolate, biopterin, and folate	Agrobacterium fabrum C58	?	-	-
additional information	enzyme PruA does not exhibit pteridine reductase activity with 7,8-dihydrofolate or fully oxidized pterins. PruA exhibits maximal catalytic efficiency with H2BPt, while both H2MPt and 7,8-dihydroneopterin (H2NPt) also serve as competent substrates. Substrate specificity, overview. No activity with 7,8-dihydrofolate, biopterin, and folate	Agrobacterium fabrum ATCC 33970	?	-	-

Subunits

Subunits	Comment	Organism
homodimer	-	Leishmania major
homodimer	2 * 28000, SDS-PAGE	Agrobacterium fabrum

Synonyms

Synonyms	Comment	Organism
Atu1130	-	Agrobacterium fabrum
More	cf. EC 1.5.1.50	Agrobacterium fabrum
PruA	-	Agrobacterium fabrum
pteridine reductase	-	Leishmania major
pteridine reductase	-	Agrobacterium fabrum
PTR1	-	Leishmania major

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Agrobacterium fabrum

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.8	-	7,8-dihydromonapterin	recombinant mutant R24A, pH 6.0, 25°C	Agrobacterium fabrum
1.4	-	7,8-dihydromonapterin	recombinant mutant Y161A, pH 6.0, 25°C	Agrobacterium fabrum
2	-	7,8-dihydromonapterin	recombinant mutant Y161F/Y163F, pH 6.0, 25°C	Agrobacterium fabrum
2.3	-	7,8-dihydromonapterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum
2.4	-	7,8-dihydroneopterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum
4.2	-	7,8-dihydromonapterin	recombinant mutant Y163F, pH 6.0, 25°C	Agrobacterium fabrum
4.7	-	7,8-dihydrobiopterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	with substrates 7,8-dihydromonapterin and NADPH	Agrobacterium fabrum

pH Range

pH Minimum	pH Maximum	Comment	Organism
4	8	the enzyme has a pH profile somewhat broader than that of other pteridine reductases and exhibits about 20% of maximal activity at pH 8.0, inactive at pH 10.0, maximal activity at pH 6.0	Agrobacterium fabrum

Cofactor

Cofactor	Comment	Organism	Structure
NADPH	-	Leishmania major
NADPH	-	Agrobacterium fabrum

General Information

General Information	Comment	Organism
evolution	the enzyme belong to the short-chain dehydrogenase/reductase (SDR) family of enzymes. Despite the overall low sequence identity among members of the SDR family (about 15-30%), a central catalytic YX3K motif is highly conserved, as is an N-terminal glycine motif (TGX3GXG), involved in cofactor binding and recognition. The pteridine reductases in the SDR family have an arginine in place of the glycine at position 6 in this motif (TGX3RXG)	Leishmania major
evolution	the enzyme belong to the short-chain dehydrogenase/reductase (SDR) family of enzymes. Despite the overall low sequence identity among members of the SDR family (about 15-30%), a central catalytic YX3K motif is highly conserved, as is an N-terminal glycine motif (TGX3GXG), involved in cofactor binding and recognition. The pteridine reductases in the SDR family have an arginine in place of the glycine at position 6 in this motif (TGX3RXG)	Agrobacterium fabrum
malfunction	the mutant DELTApruA strain exhibits elevated biofilm formation and abundant polysaccharide production independent of surface contact. The plasmid-borne expression of wild-type PruA fully corrects this deficiency, while a plasmid-encoded PruA Y163A mutant variant expressed in the DELTApruA strain has no effect on these DELTApruA mutant phenotypes, with its phenotype appearing similar to the phenotype of the deletion strain alone	Agrobacterium fabrum
additional information	enzyme structure modelling, residue Y163 is involved in catalysis	Agrobacterium fabrum
additional information	the critical proton donor in the reaction is a tyrosine residue which is part of a highly conserved YX3K motif, Tyr 194 and Lys 198 in Leishmania major PTR1	Leishmania major
physiological function	biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen Agrobacterium tumefaciens, the transition from a free-living motile state to a nonmotile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. Involvement of pterins in biofilm formation. PruA pteridine reductase is involved in the signaling pathway. The enzymatic activity of PruA is essential for the proposed pterin-dependent regulatory pathway. Wild-type Agrobacterium tumefaciens exhibits basal levels of biofilm formation in laboratory culture and forms pale orange or white colonies when grown on solid growth medium supplemented with Congo red. PruA is an important factor in controlling the motile-to-sessile transition in Agrobacterium tumefaciens	Agrobacterium fabrum
physiological function	PTR1 produces tetrahydrobiopterin (H4BPt) from dihydrobiopterin (H2BPt) or from fully oxidized biopterin (BPt) scavenged from host cells	Leishmania major

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
2.77	-	7,8-dihydromonapterin	recombinant mutant Y161A, pH 6.0, 25°C	Agrobacterium fabrum
4.72	-	7,8-dihydromonapterin	recombinant mutant R24A, pH 6.0, 25°C	Agrobacterium fabrum
6	-	7,8-dihydromonapterin	recombinant mutant Y161F/Y163F, pH 6.0, 25°C	Agrobacterium fabrum
19.75	-	7,8-dihydromonapterin	recombinant mutant Y163F, pH 6.0, 25°C	Agrobacterium fabrum
25.24	-	7,8-dihydroneopterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum
32.26	-	7,8-dihydromonapterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum
135.45	-	7,8-dihydrobiopterin	recombinant wild-type enzyme, pH 6.0, 25°C	Agrobacterium fabrum