Literature summary for 3.1.3.11 extracted from

Kuznetsova, E.; Xu, L.; Singer, A.; Brown, G.; Dong, A.; Flick, R.; Cui, H.; Cuff, M.; Joachimiak, A.; Savchenko, A.; Yakunin, A.F.
Structure and activity of the metal-independent fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae (2010), J. Biol. Chem., 285, 21049-21059.

Cloned(Commentary)

Cloned (Comment)	Organism
gene YKR043C, DNA and amino acid sequence determination and analysis, recombinant expression of the N-terminally His6-tagged enzyme, comtaining a tobacco etch virus protease cleavage site, in Escherichia coli strain BL21(DE3)	Saccharomyces cerevisiae

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified selenomethionine-substituted YK23 in apo-form, hanging drop vapor diffusion method, 22°C, purified YK23, at 29 mg/ml, is mixed with 1.5 mg/ml of trypsin solution in 1 mM HCl and 2 mM CaCl2 in a ratio of 1:10 for the apoenzyme and or 1:20 for the phosphate-bound enzyme, mixing of 0.002 ml of protein solution with 0.002 ml of reservoir solution, containing 0.2 M trilithium citrate, pH 8.1, 16% PEG 3350 and 4% 2-methyl-2,4-pentanediol, 2 days, X-ray diffraction structure determination and analysis at 1.75 A resolution, molecular replacement, modelling	Saccharomyces cerevisiae

Crystallization (Comment)

Organism

purified selenomethionine-substituted YK23 in apo-form, hanging drop vapor diffusion method, 22°C, purified YK23, at 29 mg/ml, is mixed with 1.5 mg/ml of trypsin solution in 1 mM HCl and 2 mM CaCl2 in a ratio of 1:10 for the apoenzyme and or 1:20 for the phosphate-bound enzyme, mixing of 0.002 ml of protein solution with 0.002 ml of reservoir solution, containing 0.2 M trilithium citrate, pH 8.1, 16% PEG 3350 and 4% 2-methyl-2,4-pentanediol, 2 days, X-ray diffraction structure determination and analysis at 1.75 A resolution, molecular replacement, modelling

Saccharomyces cerevisiae

Protein Variants

Protein Variants	Comment	Organism
E99A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
H13A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
H178A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
H244A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
H268A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
R181A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
R69A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
S19A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
S65A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
W131A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae
Y24A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Saccharomyces cerevisiae

Inhibitors

Inhibitors	Comment	Organism
Cd2+	-	Saccharomyces cerevisiae
Cu2+	-	Saccharomyces cerevisiae
D-fructose-2,6-bisphosphate	competitive inhibition	Saccharomyces cerevisiae
phosphate	-	Saccharomyces cerevisiae
Zn2+	-	Saccharomyces cerevisiae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	with all natural substrates, YK23 shows classical hyperbolic saturation kinetics	Saccharomyces cerevisiae
0.2	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, wild-type enzyme	Saccharomyces cerevisiae
0.3	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant R69A	Saccharomyces cerevisiae
0.5	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H268A	Saccharomyces cerevisiae
0.6	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant Y24A	Saccharomyces cerevisiae
0.7	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant S19A	Saccharomyces cerevisiae
0.9	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant S65A	Saccharomyces cerevisiae
0.9	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant W131A	Saccharomyces cerevisiae
1.4	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant R181A	Saccharomyces cerevisiae
1.5	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H178A	Saccharomyces cerevisiae
2.3	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H244A	Saccharomyces cerevisiae

Metals/Ions

Metals/Ions	Comment	Organism	Structure
additional information	the enzyme is independent of metals, Mg2+, Mn2+, Co2+, Ni2+, Li+, and Ca2+ show no effect on activity	Saccharomyces cerevisiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
D-fructose 1,6-bisphosphate + H2O	Saccharomyces cerevisiae	best substrate	D-fructose 6-phosphate + phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	P36136	gene YKR043C	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-fructose 1,6-bisphosphate + H2O	best substrate	Saccharomyces cerevisiae	D-fructose 6-phosphate + phosphate	-	?
D-fructose 1,6-bisphosphate + H2O	the substrate is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site	Saccharomyces cerevisiae	D-fructose 6-phosphate + phosphate	-	?
additional information	purified YK23 exhibits high phosphatase activity against fructose 1,6-bisphosphate, significant hydrolytic activity toward fructose 1-phosphate, and detectable activity with glyceraldehyde 3-phosphate, erythrose 4-phosphate, and ribulose 1,5-diphosphate	Saccharomyces cerevisiae	?	-	?

Subunits

Subunits	Comment	Organism
More	the core domain with the alpha/beta/alpha-fold is covered by two small cap domains, structures of presence of two phosphate molecules as inhibitor or FBP (a substrate) bound to the active site, overview	Saccharomyces cerevisiae

Synonyms

Synonyms	Comment	Organism
FBPase	-	Saccharomyces cerevisiae
fructose-1,6-bisphosphatase	-	Saccharomyces cerevisiae
More	YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily	Saccharomyces cerevisiae
YK23	-	Saccharomyces cerevisiae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Saccharomyces cerevisiae

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.3	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant S65A	Saccharomyces cerevisiae
0.5	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H178A	Saccharomyces cerevisiae
0.7	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant Y24A	Saccharomyces cerevisiae
1.6	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant R181A	Saccharomyces cerevisiae
1.8	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant R69A	Saccharomyces cerevisiae
1.9	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant W131A	Saccharomyces cerevisiae
2.8	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H244A	Saccharomyces cerevisiae
2.9	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant S19A	Saccharomyces cerevisiae
4.6	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H268A	Saccharomyces cerevisiae
4.9	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, wild-type enzyme	Saccharomyces cerevisiae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	6.5	-	Saccharomyces cerevisiae

General Information

General Information	Comment	Organism
evolution	YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily. YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes	Saccharomyces cerevisiae
physiological function	fructose-1,6-bisphosphatase is a key enzyme of gluconeogenesis and photosynthetic CO2 fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways	Saccharomyces cerevisiae

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
300	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant S65A	Saccharomyces cerevisiae
340	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H178A	Saccharomyces cerevisiae
1100	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant R181A	Saccharomyces cerevisiae
1200	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H244A	Saccharomyces cerevisiae
1200	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant Y24A	Saccharomyces cerevisiae
2200	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant W131A	Saccharomyces cerevisiae
4100	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant S19A	Saccharomyces cerevisiae
5700	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant R69A	Saccharomyces cerevisiae
9500	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, mutant H268A	Saccharomyces cerevisiae
27000	-	D-fructose 1,6-bisphosphate	pH 7.5, 30°C, wild-type enzyme	Saccharomyces cerevisiae