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Literature summary 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.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
4.2.1.11 HCO3- Km about 50% increased in presence of sodium bicarbonate, crystallized enzyme structure complexed with HCO3- in its activated form Geobacillus kaustophilus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.11 expressed in Escherichia coli BL21, pET17b expression vector Geobacillus kaustophilus
4.2.1.11 expressed in Escherichia coli, PCR-amplified genomic DNA from Bacillus subtilis strain 168 ligated into pET15b encoding an N-terminal six-His tag vector Bacillus subtilis
5.3.2.5 expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus subtilis
5.3.2.5 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Geobacillus kaustophilus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.1.11 hanging drop method, 17 A resolution, X-ray coordinates and structure factors determined, complexes with phosphate, with Mg2+, with Mg2+ and HCO3-, with Mg2+ and the alternate substrate 2,3-diketohexane 1-phosphate identified, activated enolase carboxylated on Lys173, conserved Lys98 in the N-terminal domain determined for C1 proton abstraction Geobacillus kaustophilus
5.3.2.5 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

EC Number Protein Variants Comment Organism
4.2.1.11 K147A requires a 10fold greater concentration of protein for observation of enolization Geobacillus kaustophilus
4.2.1.11 K173A detectable activity of about 3% of that of wild-type enolase, retains ability to enolize the desthio substrate Geobacillus kaustophilus
4.2.1.11 K98A unable to catalyze the enolase reaction Geobacillus kaustophilus
4.2.1.11 additional information site-directed mutagenesis of active site residues, spectrophotometric activity assay performed with elevated concentrations of the mutant enzymes Geobacillus kaustophilus
5.3.2.5 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
5.3.2.5 K173A site-directed mutagenesis, the mutant is able to catalyze enolization at approximately the same rate as the wild-type enzyme Geobacillus kaustophilus
5.3.2.5 K98A site-directed mutagenesis, the mutant is unable to catalyze the enolase reaction Geobacillus kaustophilus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.1.11 Mg2+ activity depends on Geobacillus kaustophilus
5.3.2.5 Mg2+ active site Mg2+, required Bacillus subtilis
5.3.2.5 Mg2+ active site Mg2+, required Geobacillus kaustophilus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate Geobacillus kaustophilus methionine salvage pathway 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate Bacillus subtilis methionine salvage pathway 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate Bacillus subtilis 168 methionine salvage pathway 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
5.3.2.5 5-(methylthio)-2,3-dioxopentyl phosphate Bacillus subtilis
-
 2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate
-
 ?
5.3.2.5 5-(methylthio)-2,3-dioxopentyl phosphate Geobacillus kaustophilus
-
 2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate
-
 ?
5.3.2.5 5-(methylthio)-2,3-dioxopentyl phosphate Bacillus subtilis 168
-
 2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate
-
 ?
5.3.2.5 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 ?
-
 ?
5.3.2.5 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 ?
-
 ?
5.3.2.5 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

EC Number Organism UniProt Comment Textmining
4.2.1.11 Bacillus subtilis P37869
-
-
4.2.1.11 Bacillus subtilis 168 P37869
-
-
4.2.1.11 Geobacillus kaustophilus Q5KVE7
-
-
5.3.2.5 Bacillus subtilis
-
-
-
5.3.2.5 Bacillus subtilis 168
-
-
-
5.3.2.5 Geobacillus kaustophilus Q5L1E2
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.11 recombinant protein, gel filtration Geobacillus kaustophilus
4.2.1.11 recombinant protein, gel filtration Bacillus subtilis
5.3.2.5 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Bacillus subtilis
5.3.2.5 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

EC Number Reaction Comment Organism Reaction ID
5.3.2.5 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
5.3.2.5 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

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.2.1.11 additional information
-
 spectrophotometric assay described, D-ribulose 1-phosphate and 5-methylthio-D-ribulose 1-phosphate in the presence of limiting 5-methylthio-D-ribulose 1-phosphate dehydratase analyzed, enzyme concentrations from 0.1 to 10 microM used Geobacillus kaustophilus
4.2.1.11 additional information
-
 spectrophotometric assay described, generation of reaction products determined by NMR Bacillus subtilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate methionine salvage pathway Geobacillus kaustophilus 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate methionine salvage pathway Bacillus subtilis 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate methionine salvage pathway, stereochemical course of the reaction catalyzed by enolase determined, C1 proton abstraction Geobacillus kaustophilus 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate methionine salvage pathway, stereochemical course of the reaction catalyzed by enolase determined, C1 proton abstraction Bacillus subtilis 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate methionine salvage pathway Bacillus subtilis 168 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketo-5-methylthiopentane 1-phosphate methionine salvage pathway, stereochemical course of the reaction catalyzed by enolase determined, C1 proton abstraction Bacillus subtilis 168 2-hydroxy-3-keto-5-methylthiopent-1-ene 1-phosphate
-
 ?
4.2.1.11 2,3-diketohexane 1-phosphate alternate substrate, C1 proton abstraction Geobacillus kaustophilus ?
-
 ?
4.2.1.11 2,3-diketohexane 1-phosphate alternate substrate, C1 proton abstraction Bacillus subtilis ?
-
 ?
4.2.1.11 2,3-diketohexane 1-phosphate alternate substrate, C1 proton abstraction Bacillus subtilis 168 ?
-
 ?
5.3.2.5 5-(methylthio)-2,3-dioxopentyl phosphate
-
 Bacillus subtilis 2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate
-
 ?
5.3.2.5 5-(methylthio)-2,3-dioxopentyl phosphate
-
 Geobacillus kaustophilus 2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate
-
 ?
5.3.2.5 5-(methylthio)-2,3-dioxopentyl phosphate
-
 Bacillus subtilis 168 2-hydroxy-5-(methylthio)-3-oxopent-1-enyl phosphate
-
 ?
5.3.2.5 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 ?
-
 ?
5.3.2.5 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 ?
-
 ?
5.3.2.5 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

EC Number Subunits Comment Organism
4.2.1.11 More composed of a N-terminal alpha,beta domain and a C-terminal domain consisting of eight alpha,alpha barrels, polypeptides packed as tight dimers Geobacillus kaustophilus
5.3.2.5 dimer
-
 Bacillus subtilis
5.3.2.5 dimer structure modeling, overview Geobacillus kaustophilus

Synonyms

EC Number Synonyms Comment Organism
4.2.1.11 enolase
-
 Geobacillus kaustophilus
4.2.1.11 enolase
-
 Bacillus subtilis
5.3.2.5 2,3-diketo-5-methylthiopentane 1-phosphate enolase
-
 Bacillus subtilis
5.3.2.5 2,3-diketo-5-methylthiopentane 1-phosphate enolase
-
 Geobacillus kaustophilus
5.3.2.5 DK-MTP 1-P enolase
-
 Bacillus subtilis
5.3.2.5 DK-MTP 1-P enolase
-
 Geobacillus kaustophilus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.11 7.5
-
 assay at Geobacillus kaustophilus

General Information

EC Number General Information Comment Organism
5.3.2.5 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
5.3.2.5 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
5.3.2.5 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
5.3.2.5 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
5.3.2.5 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
5.3.2.5 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