Literature summary for 4.2.3.19 extracted from

Liu, W.; Feng, X.; Zheng, Y.; Huang, C.H.; Nakano, C.; Hoshino, T.; Bogue, S.; Ko, T.P.; Chen, C.C.; Cui, Y.; Li, J.; Wang, I.; Hsu, S.T.; Oldfield, E.; Guo, R.T.
Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium japonicum (2014), Sci. Rep., 4, 6214 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of thioredoxin- and His-tagged wild-type and mutant enzymes with or without selenium-methionine labeling in Escherichia coli strain BL21trxB (DE3)	Bradyrhizobium diazoefficiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type enzyme in apo form or in complex with inhibitor BPH-629, mutant D75C in complex with substrate ent-copalyl diphosphate, sitting drop vapor diffusion method, mixing of 6.4 mg/ml protein in 25 mM Tris, pH 7.5, and 150 mM NaCl with reservoir solution containing 0.7 M ammonium tartrate dibasic, pH 7.0 and 2-4% w/v PEG 4000 for the wild-type enzyme, for the inhibitor BPH-629 complex, the enzyme solution also contains 1 mM MgCl2, 10 mM DTT, and 2.5 mM BPH-629, for the substrate complex, the D75C mutant crystal is prepared under the same crystallization conditions, then soaked with the cryo-protectant containing 5 mM ent-copalyl diphosphate for 5 hours, 2-3 days, room temperature, X-ray diffraction structure determination and analysis at 1.80-2.34 A resolution	Bradyrhizobium diazoefficiens

Crystallization (Comment)

Organism

purified recombinant wild-type enzyme in apo form or in complex with inhibitor BPH-629, mutant D75C in complex with substrate ent-copalyl diphosphate, sitting drop vapor diffusion method, mixing of 6.4 mg/ml protein in 25 mM Tris, pH 7.5, and 150 mM NaCl with reservoir solution containing 0.7 M ammonium tartrate dibasic, pH 7.0 and 2-4% w/v PEG 4000 for the wild-type enzyme, for the inhibitor BPH-629 complex, the enzyme solution also contains 1 mM MgCl2, 10 mM DTT, and 2.5 mM BPH-629, for the substrate complex, the D75C mutant crystal is prepared under the same crystallization conditions, then soaked with the cryo-protectant containing 5 mM ent-copalyl diphosphate for 5 hours, 2-3 days, room temperature, X-ray diffraction structure determination and analysis at 1.80-2.34 A resolution

Bradyrhizobium diazoefficiens

Protein Variants

Protein Variants	Comment	Organism
D75A	site-directed mutagenesis	Bradyrhizobium diazoefficiens
D75C	site-directed mutagenesis, mutant crystal structure determination and analysis	Bradyrhizobium diazoefficiens
D79C	site-directed mutagenesis	Bradyrhizobium diazoefficiens
R204A	site-directed mutagenesis	Bradyrhizobium diazoefficiens

Inhibitors

Inhibitors	Comment	Organism	Structure
BPH-629	the bisphosphonate inhibitor binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues, enzyme binding structure analysis, overview	Bradyrhizobium diazoefficiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ent-copalyl diphosphate	Bradyrhizobium diazoefficiens	-	ent-kaur-16-ene + diphosphate	-	?
ent-copalyl diphosphate	Bradyrhizobium diazoefficiens JCM 10833 / IAM 13628 / NBRC 14792 / USDA 110	-	ent-kaur-16-ene + diphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Bradyrhizobium diazoefficiens	Q45222	-	-
Bradyrhizobium diazoefficiens JCM 10833 / IAM 13628 / NBRC 14792 / USDA 110	Q45222	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant thioredoxin- and His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21trxB (DE3) by nickel affinity chromatography, followed by tag cleavage by TEV protease, another step of nickel affinity chromatography, anion exchange chromatography, and ultrafiltration	Bradyrhizobium diazoefficiens

Reaction

Reaction	Comment	Organism	Reaction ID
ent-copalyl diphosphate = ent-kaurene + diphosphate	reaction mechanism, overview	Bradyrhizobium diazoefficiens	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ent-copalyl diphosphate	-	Bradyrhizobium diazoefficiens	ent-kaur-16-ene + diphosphate	-	?
ent-copalyl diphosphate	-	Bradyrhizobium diazoefficiens JCM 10833 / IAM 13628 / NBRC 14792 / USDA 110	ent-kaur-16-ene + diphosphate	-	?
additional information	the ent-copalyl diphosphate substrate binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues	Bradyrhizobium diazoefficiens	?	-	?
additional information	the ent-copalyl diphosphate substrate binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues	Bradyrhizobium diazoefficiens JCM 10833 / IAM 13628 / NBRC 14792 / USDA 110	?	-	?

Subunits

Subunits	Comment	Organism
More	enzyme structure comparisons, overview	Bradyrhizobium diazoefficiens

Synonyms

Synonyms	Comment	Organism
BjKS	-	Bradyrhizobium diazoefficiens
blr2150	-	Bradyrhizobium diazoefficiens
ent-kaur-16-ene synthase	-	Bradyrhizobium diazoefficiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Bradyrhizobium diazoefficiens

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
0.0095	-	pH 7.5, temperature not specified in the publication	Bradyrhizobium diazoefficiens	BPH-629

General Information

General Information	Comment	Organism
evolution	the enzyme is a class I bacterial diterpene cyclase	Bradyrhizobium diazoefficiens
metabolism	class I bacterial diterpene cyclase, ent-kaurene synthase (BjKS) catalyzes the cyclization of ent-copalyl diphosphate (ent-CPP), which is produced by the separate (class II) enzyme, ent-copalyl diphosphate synthase (ent-CPPS, EC 5.5.1.13) from geranylgeranyl diphosphate, to ent-kaurene	Bradyrhizobium diazoefficiens
additional information	the enzyme contains the highly conserved DDXXD sequence	Bradyrhizobium diazoefficiens
physiological function	ent-copalyl diphosphate (ent-CPP) is produced by the separate (class II) enzyme, ent-copalyl diphosphate synthase (ent-CPPS, EC 5.5.1.13), from geranylgeranyl diphosphate. The ent-kaur-16-ene synthase then catalyzes the cyclization of ent-copalyl diphosphate (ent-CPP) to ent-kaurene	Bradyrhizobium diazoefficiens