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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.3.7.11 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

EC Number Protein Variants Comment Organism
1.3.7.11 F219L site-directed mutagenesis, the mutant enzymes ceases GGPP reduction at H4GGPP without significant conversion to H6GGPP Sulfolobus acidocaldarius
1.3.7.11 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
1.3.7.11 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
1.3.7.11 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
1.3.7.11 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
1.3.7.11 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

EC Number Organism UniProt Comment Textmining
1.3.7.11 Sulfolobus acidocaldarius Q4JA33
-
-
1.3.7.11 Sulfolobus acidocaldarius DSM 639 Q4JA33
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.7.11 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.7.11 geranylgeranyl diphosphate + reduced dithionite non-native substrate Sulfolobus acidocaldarius hexahydro-geranylgeranyl diphoshate + oxidized dithionite
-
 ?
1.3.7.11 geranylgeranyl diphosphate + reduced dithionite non-native substrate Sulfolobus acidocaldarius DSM 639 hexahydro-geranylgeranyl diphoshate + oxidized dithionite
-
 ?
1.3.7.11 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 ?
-
 ?
1.3.7.11 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

EC Number Synonyms Comment Organism
1.3.7.11 geranylgeranyl reductase
-
 Sulfolobus acidocaldarius
1.3.7.11 GGR
-
 Sulfolobus acidocaldarius
1.3.7.11 SaGGR
-
 Sulfolobus acidocaldarius

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.3.7.11 50
-
 assay at Sulfolobus acidocaldarius

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.3.7.11 0.0067
-
 geranylgeranyl diphosphate wild-type enzyme, pH 5.5, 50°C Sulfolobus acidocaldarius
1.3.7.11 0.0088
-
 geranylgeranyl diphosphate mutant I206F, pH 5.5, 50°C Sulfolobus acidocaldarius
1.3.7.11 0.013
-
 geranylgeranyl diphosphate mutant F219L, pH 5.5, 50°C Sulfolobus acidocaldarius
1.3.7.11 0.0138
-
 geranylgeranyl diphosphate mutant G91H, pH 5.5, 50°C Sulfolobus acidocaldarius
1.3.7.11 0.0156
-
 geranylgeranyl diphosphate mutant L377H, pH 5.5, 50°C Sulfolobus acidocaldarius
1.3.7.11 0.0158
-
 geranylgeranyl diphosphate mutant I206F/L377H, pH 5.5, 50°C Sulfolobus acidocaldarius

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.3.7.11 5.5
-
 assay at Sulfolobus acidocaldarius

Cofactor

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