Literature summary for 1.3.7.11 extracted from

Kung, Y.; McAndrew, R.P.; Xie, X.; Liu, C.C.; Pereira, J.H.; Adams, P.D.; Keasling, J.D.
Constructing tailored isoprenoid products by structure-guided modification of geranylgeranyl reductase (2014), Structure, 22, 1028-1036 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strains BLR(DE3) or Rosetta2(DE3)	Sulfolobus acidocaldarius

Crystallization (Commentary)

Crystallization (Comment)	Organism
wild-type and mutant enzymes, in apoform or complexed with geranygeranyl diphosphate, sitting drop vapor diffusion, mixing of 10 mg/ml protein in 25 mM HEPES, pH 7.4, with well solution containing 0.1M Tris, pH 7.5, 10% PEG 3350, and 0.2 M L-proline, in a 1:1 ratio, sparse matrix-screening crystallization method for method screening, addition of 5 mM GGPP for ligand-bound crystals, 2 days, X-ray diffractin structure determination and analysis, modeling	Sulfolobus acidocaldarius

Crystallization (Comment)

Organism

wild-type and mutant enzymes, in apoform or complexed with geranygeranyl diphosphate, sitting drop vapor diffusion, mixing of 10 mg/ml protein in 25 mM HEPES, pH 7.4, with well solution containing 0.1M Tris, pH 7.5, 10% PEG 3350, and 0.2 M L-proline, in a 1:1 ratio, sparse matrix-screening crystallization method for method screening, addition of 5 mM GGPP for ligand-bound crystals, 2 days, X-ray diffractin structure determination and analysis, modeling

Sulfolobus acidocaldarius

Protein Variants

Protein Variants	Comment	Organism
F219L	site-directed mutagenesis, the mutant enzymes ceases GGPP reduction at H4GGPP without significant conversion to H6GGPP	Sulfolobus acidocaldarius
G91H	site-directed mutagenesis, the mutant stops the reduction of GGPP at H2GGPP, only a very small quantity of H2GGPP is reduced further to H4GGPP or to H6GGPP	Sulfolobus acidocaldarius
I206F	site-directed mutagenesis, the mutant does not accumulate appreciable levels of H2GGPP at any point during the reaction, the mutant enzyme shows increased H6GGPP production compared to the wild-type enzyme	Sulfolobus acidocaldarius
I206F/L377H	site-directed mutagenesis, the mutant does not accumulate appreciable levels of H2GGPP at any point during the reaction, the mutant enzyme shows increased H6GGPP production compared to the wild-type enzyme	Sulfolobus acidocaldarius
L377H	site-directed mutagenesis, the mutant does not accumulate appreciable levels of H2GGPP at any point during the reaction, the mutant enzyme shows increased H6GGPP production compared to the wild-type enzyme	Sulfolobus acidocaldarius
additional information	structure-guided design of the enzyme yields SaGGR variants that enhance the rate of H6GGPP product formation. Additional mutants are observed to arrest the degree of GGPP reduction at H2GGPP and H4GGPP. Crystal structures of these variants reveal the structural bases for their altered activities, in addition to providing insight into the SaGGR mechanism. Three mutants (I206F, L377H, and I206F/L377H) exhibit faster production of H6GGPP than the wild-type, increasing the overall rate of product formation by up to 2.4fold	Sulfolobus acidocaldarius

Organism

Organism	UniProt	Comment	Textmining
Sulfolobus acidocaldarius	Q4JA33	-	-
Sulfolobus acidocaldarius DSM 639	Q4JA33	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strains BLR(DE3) or Rosetta2(DE3) by nickel affinity chromatography, cleavage of the His-tag by TEV protease, followed by another step of nickel affinity chromatography to remove the tag, followed by desalting gel filtration	Sulfolobus acidocaldarius

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
geranylgeranyl diphosphate + reduced dithionite	non-native substrate	Sulfolobus acidocaldarius	hexahydro-geranylgeranyl diphoshate + oxidized dithionite	-	?
geranylgeranyl diphosphate + reduced dithionite	non-native substrate	Sulfolobus acidocaldarius DSM 639	hexahydro-geranylgeranyl diphoshate + oxidized dithionite	-	?
additional information	the wild-type enzyme reduces the non-native substrate geranylgeranyl diphoshate with reductant sodium dithionite and produces H6GGPP. Geranylgeranyl diphosphate is bound by three binding sites for GGPP, mechansim of the enzyme orientating double bonds to be reduced by the bound FAD cofactor, overview. Catalytic reaction mechansim and substrate binding structure, overview	Sulfolobus acidocaldarius	?	-	?
additional information	the wild-type enzyme reduces the non-native substrate geranylgeranyl diphoshate with reductant sodium dithionite and produces H6GGPP. Geranylgeranyl diphosphate is bound by three binding sites for GGPP, mechansim of the enzyme orientating double bonds to be reduced by the bound FAD cofactor, overview. Catalytic reaction mechansim and substrate binding structure, overview	Sulfolobus acidocaldarius DSM 639	?	-	?

Synonyms

Synonyms	Comment	Organism
geranylgeranyl reductase	-	Sulfolobus acidocaldarius
GGR	-	Sulfolobus acidocaldarius
SaGGR	-	Sulfolobus acidocaldarius

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
50	-	assay at	Sulfolobus acidocaldarius

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0067	-	geranylgeranyl diphosphate	wild-type enzyme, pH 5.5, 50°C	Sulfolobus acidocaldarius
0.0088	-	geranylgeranyl diphosphate	mutant I206F, pH 5.5, 50°C	Sulfolobus acidocaldarius
0.013	-	geranylgeranyl diphosphate	mutant F219L, pH 5.5, 50°C	Sulfolobus acidocaldarius
0.0138	-	geranylgeranyl diphosphate	mutant G91H, pH 5.5, 50°C	Sulfolobus acidocaldarius
0.0156	-	geranylgeranyl diphosphate	mutant L377H, pH 5.5, 50°C	Sulfolobus acidocaldarius
0.0158	-	geranylgeranyl diphosphate	mutant I206F/L377H, pH 5.5, 50°C	Sulfolobus acidocaldarius

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5.5	-	assay at	Sulfolobus acidocaldarius

Cofactor

Cofactor	Comment	Organism	Structure
FAD	the GGPP DELTA14 double bond is directly adjacent to the N5 position of the FAD cofactor, binding structure, overview	Sulfolobus acidocaldarius
additional information	no activity with NAD(P)H	Sulfolobus acidocaldarius