Literature summary for 5.3.2.5 extracted from

Imker, H.J.; Fedorov, A.A.; Fedorov, E.V.; Almo, S.C.; Gerlt, J.A.
Mechanistic diversity in the RuBisCO superfamily: the enolase in the methionine salvage pathway in Geobacillus kaustophilus (2007), Biochemistry, 46, 4077-4089.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Bacillus subtilis
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Geobacillus kaustophilus

Crystallization (Commentary)

Crystallization (Comment)	Organism
activated wild-type enolase, carboxylated on Lys173, with Mg2+ alone, or Mg2+, and HCO3-, or Mg2+ and substrate 2,3-diketohexane 1-phosphate, and of the selenomethionine-substituted enzyme variant, hanging drop method, 10-15 mg/ml protein in 20 mM Tris-HCl, pH 7.9, 100 mM NaCl, and 5-10 mM MgCl2, 100 mM NaCl, and 1-5 mM NaHCO3, mixed with 24% PEG 3350, 0.1 M Tris-HCl, pH 8.5, and 0.2 M ammonium acetate as precipitant for the crystals of wild-type enzyme with Mg2+, or 25% PEG 3350, 0.1 M HEPES, pH 7.5, and 0.2 M ammonium acetate as precipitant for the other crystal variants, room temperature, 6 days, X-ray diffraction structure determination and analysis at 1.7 A resolution, molecular replacement	Geobacillus kaustophilus

Crystallization (Comment)

Organism

activated wild-type enolase, carboxylated on Lys173, with Mg2+ alone, or Mg2+, and HCO3-, or Mg2+ and substrate 2,3-diketohexane 1-phosphate, and of the selenomethionine-substituted enzyme variant, hanging drop method, 10-15 mg/ml protein in 20 mM Tris-HCl, pH 7.9, 100 mM NaCl, and 5-10 mM MgCl2, 100 mM NaCl, and 1-5 mM NaHCO3, mixed with 24% PEG 3350, 0.1 M Tris-HCl, pH 8.5, and 0.2 M ammonium acetate as precipitant for the crystals of wild-type enzyme with Mg2+, or 25% PEG 3350, 0.1 M HEPES, pH 7.5, and 0.2 M ammonium acetate as precipitant for the other crystal variants, room temperature, 6 days, X-ray diffraction structure determination and analysis at 1.7 A resolution, molecular replacement

Geobacillus kaustophilus

Protein Variants

Protein Variants	Comment	Organism
K147A	site-directed mutagenesis, the mutant requires a 10fold greater concentration of protein for enolization of the natural substrate, reduced activity compared to the wild-type enzyme	Geobacillus kaustophilus
K173A	site-directed mutagenesis, the mutant is able to catalyze enolization at approximately the same rate as the wild-type enzyme	Geobacillus kaustophilus
K98A	site-directed mutagenesis, the mutant is unable to catalyze the enolase reaction	Geobacillus kaustophilus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	active site Mg2+, required	Bacillus subtilis
Mg2+	active site Mg2+, required	Geobacillus kaustophilus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
5-(methylthio)-2,3-dioxopentyl phosphate	Bacillus subtilis	-	2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate	-	?
5-(methylthio)-2,3-dioxopentyl phosphate	Geobacillus kaustophilus	-	2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate	-	?
5-(methylthio)-2,3-dioxopentyl phosphate	Bacillus subtilis 168	-	2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate	-	?
additional information	Bacillus subtilis	the enzyme catalyzes the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine. The reaction is accomplished by abstraction of the 1-proS proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Stabilization of an enolate anion intermediate by coordination to an active site Mg2+. Structure-function relationships, overview. The enolase from Geobacillus kaustophilus catalyzes stereospecific abstraction of the 1-proS proton of the DK-MTP 1-P substrate	?	-	?
additional information	Geobacillus kaustophilus	the enzyme catalyzes the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine. The reaction is accomplished by abstraction of the 1-proS proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Stabilization of an enolate anion intermediate by coordination to an active site Mg2+. Structure-function relationships, overview. The enolase from Geobacillus kaustophilus catalyzes stereospecific abstraction of the 1-proS proton of the DK-MTP 1-P substrate	?	-	?
additional information	Bacillus subtilis 168	the enzyme catalyzes the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine. The reaction is accomplished by abstraction of the 1-proS proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Stabilization of an enolate anion intermediate by coordination to an active site Mg2+. Structure-function relationships, overview. The enolase from Geobacillus kaustophilus catalyzes stereospecific abstraction of the 1-proS proton of the DK-MTP 1-P substrate	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Bacillus subtilis	-	-	-
Bacillus subtilis 168	-	-	-
Geobacillus kaustophilus	Q5L1E2	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Bacillus subtilis
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by two different steps of anion exchange chromatography and ultrafiltration	Geobacillus kaustophilus

Reaction

Reaction	Comment	Organism	Reaction ID
5-(methylsulfanyl)-2,3-dioxopentyl phosphate = 2-hydroxy-5-(methylsulfanyl)-3-oxopent-1-enyl phosphate	structure-function relationship and reaction mechanism, overview	Bacillus subtilis	a
5-(methylsulfanyl)-2,3-dioxopentyl phosphate = 2-hydroxy-5-(methylsulfanyl)-3-oxopent-1-enyl phosphate	structure-function relationship and reaction mechanism, overview	Geobacillus kaustophilus	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
5-(methylthio)-2,3-dioxopentyl phosphate	-	Bacillus subtilis	2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate	-	?
5-(methylthio)-2,3-dioxopentyl phosphate	-	Geobacillus kaustophilus	2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate	-	?
5-(methylthio)-2,3-dioxopentyl phosphate	-	Bacillus subtilis 168	2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate	-	?
additional information	the enzyme catalyzes the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine. The reaction is accomplished by abstraction of the 1-proS proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Stabilization of an enolate anion intermediate by coordination to an active site Mg2+. Structure-function relationships, overview. The enolase from Geobacillus kaustophilus catalyzes stereospecific abstraction of the 1-proS proton of the DK-MTP 1-P substrate	Bacillus subtilis	?	-	?
additional information	the enzyme catalyzes the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine. The reaction is accomplished by abstraction of the 1-proS proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Stabilization of an enolate anion intermediate by coordination to an active site Mg2+. Structure-function relationships, overview. The enolase from Geobacillus kaustophilus catalyzes stereospecific abstraction of the 1-proS proton of the DK-MTP 1-P substrate	Geobacillus kaustophilus	?	-	?
additional information	the enzyme catalyzes the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine. The reaction is accomplished by abstraction of the 1-proS proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Stabilization of an enolate anion intermediate by coordination to an active site Mg2+. Structure-function relationships, overview. The enolase from Geobacillus kaustophilus catalyzes stereospecific abstraction of the 1-proS proton of the DK-MTP 1-P substrate	Bacillus subtilis 168	?	-	?

Subunits

Subunits	Comment	Organism
dimer	-	Bacillus subtilis
dimer	structure modeling, overview	Geobacillus kaustophilus

Synonyms

Synonyms	Comment	Organism
2,3-diketo-5-methylthiopentane 1-phosphate enolase	-	Bacillus subtilis
2,3-diketo-5-methylthiopentane 1-phosphate enolase	-	Geobacillus kaustophilus
DK-MTP 1-P enolase	-	Bacillus subtilis
DK-MTP 1-P enolase	-	Geobacillus kaustophilus

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the RuBisCO superfamily. The functionally divergent Geobacillus kaustophilus member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base	Bacillus subtilis
evolution	the enzyme belongs to the RuBisCO superfamily. The functionally divergent Geobacillus kaustophilus member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base	Geobacillus kaustophilus
metabolism	the enzyme is involved in the methionine salvage pathway. It is a heterofunctional homologue of RuBisCO catalyzing the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine	Bacillus subtilis
metabolism	the enzyme is involved in the methionine salvage pathway. It is a heterofunctional homologue of RuBisCO catalyzing the tautomerization of 2,3-diketo-5-methylthiopentane 1-phosphate in the methionine salvage pathway in which 5-methylthio-D-ribose derived from 5'-methylthioadenosine is converted to methionine	Geobacillus kaustophilus
additional information	structure-function relationship, the active site KDDE motif with the carboxylated Lys173 as well as two carboxylate ligands Asp175 and Glu176 are essential for Mg2+ stabilization and also for stabilizing the enediolate intermediate, the carbamate group of the carboxylated Lys173 is the third ligand for the essential Mg2+ as well as the general base Lys98 that initiates the reaction by abstraction of the proton from the substrate	Bacillus subtilis
additional information	structure-function relationship, the active site KDDE motif with the carboxylated Lys173 as well as two carboxylate ligands Asp175 and Glu176 are essential for Mg2+ stabilization and also for stabilizing the enediolate intermediate, the carbamate group of the carboxylated Lys173 is the third ligand for the essential Mg2+ as well as the general base Lys98 that initiates the reaction by abstraction of the proton from the substrate	Geobacillus kaustophilus