Literature summary for 1.17.7.4 extracted from

Graewert, T.; Span, I.; Eisenreich, W.; Rohdich, F.; Eppinger, J.; Bacher, A.; Groll, M.
Probing the reaction mechanism of IspH protein by x-ray structure analysis (2010), Proc. Natl. Acad. Sci. USA, 107, 1077-1081.

Cloned(Commentary)

Cloned (Comment)	Organism
cloning from genomic DNA and expression as His-tagged protein in Escherichia coli strain XL-1 Blue	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant IspH, sitting drop vapour diffusion method at 20°C, 0.004 ml of 35 /ml protein in 50 mM Tris-HCl, pH 8.0, is mixed with 0.004 ml of reservoir solution containing 100 mM Bis-Tris-HCl, pH 6.5, 200 mM lithium sulfate and 25% PEG 3350, final pH is 6.8, crystal soaking in 2 M lithium sulfate, pH 6.5, before freezing, X-ray diffraction structure determination and analysis at 1.4 A resolution, molecular replacement, modelling	Escherichia coli

Crystallization (Comment)

Organism

purified recombinant IspH, sitting drop vapour diffusion method at 20°C, 0.004 ml of 35 /ml protein in 50 mM Tris-HCl, pH 8.0, is mixed with 0.004 ml of reservoir solution containing 100 mM Bis-Tris-HCl, pH 6.5, 200 mM lithium sulfate and 25% PEG 3350, final pH is 6.8, crystal soaking in 2 M lithium sulfate, pH 6.5, before freezing, X-ray diffraction structure determination and analysis at 1.4 A resolution, molecular replacement, modelling

Escherichia coli

General Stability

General Stability	Organism
IspH is more stable in its closed conformation with a substrate molecule bound to the cluster, compared to its open conformation in solution	Escherichia coli

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	IspH contains a [4Fe-4S] cluster	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H + H+	Escherichia coli	in the nonmevalonate pathway, the terminal intermediate, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, i.e. HMBPP, is reductively converted into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate	dimethylallyl diphosphate + NAD(P)+ + H2O	-	r
(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H + H+	Escherichia coli	in the nonmevalonate pathway, the terminal intermediate, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, i.e. HMBPP, is reductively converted into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate	isopentenyl diphosphate + NAD(P)+ + H2O	-	r

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	-	gene ispH, formerly lytB	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged IspH from strain XL-1 Blue by nickel affinity and anion exchange chromatography followed by dialysis, over 99% purity	Escherichia coli

Reaction

Reaction	Comment	Organism	Reaction ID
dimethylallyl diphosphate + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O = (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	reaction mechanism, detailed overview	Escherichia coli	a
isopentenyl diphosphate + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O = (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	reaction mechanism, detailed overview	Escherichia coli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H + H+	in the nonmevalonate pathway, the terminal intermediate, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, i.e. HMBPP, is reductively converted into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate	Escherichia coli	dimethylallyl diphosphate + NAD(P)+ + H2O	-	r
(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H + H+	formation of a mixture of isopentenyl diphosphate and dimethylallyl diphosphate	Escherichia coli	dimethylallyl diphosphate + NAD(P)+ + H2O	-	r
(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H + H+	in the nonmevalonate pathway, the terminal intermediate, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, i.e. HMBPP, is reductively converted into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate	Escherichia coli	isopentenyl diphosphate + NAD(P)+ + H2O	-	r
(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H + H+	formation of a mixture of isopentenyl diphosphate and dimethylallyl diphosphate	Escherichia coli	isopentenyl diphosphate + NAD(P)+ + H2O	-	r
additional information	active site structure analysis of the IspH protein in complex with the substrate and the reaction products, or with diphosphate, overview. IspH is more stable in its closed conformation with a substrate molecule bound to the cluster, compared to its open conformation in solution	Escherichia coli	?	-	?

Subunits

Subunits	Comment	Organism
monomer	-	Escherichia coli

Synonyms

Synonyms	Comment	Organism
ispH	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Escherichia coli