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Literature summary extracted from

  • Rakus, J.F.; Fedorov, A.A.; Fedorov, E.V.; Glasner, M.E.; Hubbard, B.K.; Delli, J.D.; Babbitt, P.C.; Almo, S.C.; Gerlt, J.A.
    Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase (2008), Biochemistry, 47, 9944-9954.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.1.2.53 expression in Escherichia coli BL21(DE3) Escherichia coli
4.2.1.90 gene yfaW, expression of the His6- or His10-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Escherichia coli K-12

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.1.90 enzyme bound to Mg2+ and 3-deoxy-L-rhamnonate, hanging drop method at room temperature, the protein solution for the Mg2+-enzyme complex contains 9.7 mg/mL RhamD in 10 mM HEPES, pH 7.5, containing 150 mM NaCl, 10 mM L-methionine, 5 mM DTT, 10% glycerol, and 5 mM MgCl2, the precipitant solution contains 2.4 M sodium malonate, pH 7.0, and 5 mM MgCl2, for the tertiary complex the protein solution contains 42 mg/mL RhamD in 10 mM in HEPES, pH 7.5, 150 mm NaCl, 10 mM L-methionine, 5 mM DTT, 10% glycerol, 5 mM MgCl2, and 40 mM 3-deoxy-L-rhamnonate, the precipitant solution contains 60% Tacsimate, pH 7.0, and 5 mM MgCl2, 3-4 days, X-ray diffraction structure determination and analysis at 1.8-2.0 A resolution, RhamD crystallizes as an octamer Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 enzyme bound to Mg2+, the protein solution contains 42 mg/mL RhamD in 10 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM L-methionine, and 10% glycerol, the precipitant solution contains 1.2 M ammonium sulfate, 100 mM Tris-HCl, pH 8.5, and 0.2 M lithium sulfate, 2 days, X-ray diffraction structure determination and analysis at 2.1 A resolution, RhamD crystallizes as an octamer Escherichia coli K-12

Protein Variants

EC Number Protein Variants Comment Organism
4.2.1.90 H281N site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli K-12
4.2.1.90 H282N site-directed mutagenesis Escherichia coli K-12
4.2.1.90 H329N site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli K-12
4.2.1.90 H33N site-directed mutagenesis, inactive mutant Escherichia coli K-12

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.1.2.53 0.078
-
 2-dehydro-3-deoxy-L-rhamnonate pH 8.0, 25°C Escherichia coli
4.1.2.53 0.14
-
 2-dehydro-3-deoxy-L-mannonate pH 8.0, 25°C Escherichia coli
4.1.2.53 0.8
-
 2-dehydro-3-deoxy-L-lyxonate pH 8.0, 25°C Escherichia coli
4.2.1.90 0.06
-
 L-mannonate pH 7.9, 25°C, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 0.15
-
 L-Rhamnonate pH 7.9, 25°C, wild-type enzyme Escherichia coli K-12
4.2.1.90 0.15
-
 L-mannonate pH 7.9, 25°C, wild-type enzyme Escherichia coli K-12
4.2.1.90 0.25
-
 L-Rhamnonate pH 7.9, 25°C, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 1.6
-
 L-lyxonate pH 7.9, 25°C, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 2
-
 L-lyxonate pH 7.9, 25°C, wild-type enzyme Escherichia coli K-12

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.1.2.53 Mg2+ required Escherichia coli
4.2.1.90 Mg2+
-
 Escherichia coli K-12
4.2.1.90 Mg2+
-
 Salmonella enterica subsp. enterica serovar Typhimurium

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.2.1.90 additional information
-
 RhamD crystallizes as an octamer, in agreement with the oligomeric structure determined by gel filtration Escherichia coli K-12

Organism

EC Number Organism UniProt Comment Textmining
4.1.2.53 Escherichia coli P76469
-
-
4.2.1.90 Escherichia coli K-12 P77215 gene yfaW, strain MG1655
-
4.2.1.90 Salmonella enterica subsp. enterica serovar Typhimurium Q8ZNF9
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.1.2.53
-
 Escherichia coli

Reaction

EC Number Reaction Comment Organism Reaction ID
4.2.1.90 L-rhamnonate = 2-dehydro-3-deoxy-L-rhamnonate + H2O reaction mechanism of the RhamD-catalyzed reaction, overview Escherichia coli K-12
a
4.2.1.90 L-rhamnonate = 2-dehydro-3-deoxy-L-rhamnonate + H2O reaction mechanism of the RhamD-catalyzed reaction, overview Salmonella enterica subsp. enterica serovar Typhimurium
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.1.2.53 2-dehydro-3-deoxy-L-lyxonate
-
 Escherichia coli ?
-
 ?
4.1.2.53 2-dehydro-3-deoxy-L-mannonate
-
 Escherichia coli ?
-
 ?
4.1.2.53 2-dehydro-3-deoxy-L-rhamnonate 2-dehydro-3-deoxy-L-rhamnonate i.e. 3,6-dideoxy-L-erythro-hex-2-ulosonate Escherichia coli pyruvate + (R)-lactaldehyde
-
 ?
4.2.1.90 D-gulonate low activity Escherichia coli K-12 ? + H2O
-
 ?
4.2.1.90 L-lyxonate
-
 Escherichia coli K-12 ? + H2O
-
 ?
4.2.1.90 L-lyxonate
-
 Salmonella enterica subsp. enterica serovar Typhimurium ? + H2O
-
 ?
4.2.1.90 L-mannonate
-
 Escherichia coli K-12 ? + H2O
-
 ?
4.2.1.90 L-mannonate
-
 Salmonella enterica subsp. enterica serovar Typhimurium ? + H2O
-
 ?
4.2.1.90 L-rhamnonate i.e. 6-deoxy-L-mannonate Escherichia coli K-12 2-dehydro-3-deoxy-L-rhamnonate + H2O
-
 ?
4.2.1.90 L-rhamnonate i.e. 6-deoxy-L-mannonate Salmonella enterica subsp. enterica serovar Typhimurium 2-dehydro-3-deoxy-L-rhamnonate + H2O
-
 ?
4.2.1.90 additional information stereochemical course of the RhamD-catalyzed reaction, overview Escherichia coli K-12 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
4.2.1.90 More RhamD contains an N-terminal alpha+beta capping domain and a C-terminal (beta/alpha)7beta-barrel, i.e. a modified TIM-barrel, catalytic domain with the active site located at the interface between the two domains Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 octamer RhamD contains an N-terminal alpha+beta capping domain and a C-terminal (beta/alpha)7beta-barrel, i.e. a modified TIM-barrel, catalytic domain with the active site located at the interface between the two domains Escherichia coli K-12

Synonyms

EC Number Synonyms Comment Organism
4.1.2.53 2-dehydro-3-deoxyrhamnonate aldolase
-
 Escherichia coli
4.1.2.53 2-keto-3-deoxy acid sugar aldolase
-
 Escherichia coli
4.1.2.53 KDR aldolase
-
 Escherichia coli
4.1.2.53 YfaU
-
 Escherichia coli
4.2.1.90 More the enzyme belongs to the enolase superfamily of enzymes Escherichia coli K-12
4.2.1.90 More the enzyme belongs to the enolase superfamily of enzymes Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 RhamD
-
 Escherichia coli K-12
4.2.1.90 RhamD
-
 Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 YfaW
-
 Escherichia coli K-12

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.1.2.53 25
-
 assay at Escherichia coli
4.2.1.90 25
-
 assay at Escherichia coli K-12
4.2.1.90 25
-
 assay at Salmonella enterica subsp. enterica serovar Typhimurium

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.1.2.53 0.3
-
 2-dehydro-3-deoxy-L-rhamnonate pH 8.0, 25°C Escherichia coli
4.1.2.53 0.3
-
 2-dehydro-3-deoxy-L-lyxonate pH 8.0, 25°C Escherichia coli
4.1.2.53 0.4
-
 2-dehydro-3-deoxy-L-rhamnonate pH 8.0, 25°C Escherichia coli
4.2.1.90 0.02
-
 D-gulonate pH 7.9, 25°C, wild-type enzyme Escherichia coli K-12
4.2.1.90 0.05
-
 L-lyxonate pH 7.9, 25°C, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 0.1
-
 L-mannonate pH 7.9, 25°C, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
4.2.1.90 0.2
-
 L-mannonate pH 7.9, 25°C, wild-type enzyme Escherichia coli K-12
4.2.1.90 2
-
 L-lyxonate pH 7.9, 25°C, wild-type enzyme Escherichia coli K-12
4.2.1.90 3.2
-
 L-Rhamnonate pH 7.9, 25°C, wild-type enzyme Escherichia coli K-12
4.2.1.90 3.9
-
 L-Rhamnonate pH 7.9, 25°C, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.1.2.53 8
-
 assay at Escherichia coli
4.2.1.90 7.9
-
 assay at Escherichia coli K-12
4.2.1.90 7.9
-
 assay at Salmonella enterica subsp. enterica serovar Typhimurium

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.1.2.53 2.1
-
 2-dehydro-3-deoxy-L-mannonate pH 8.0, 25°C Escherichia coli
4.1.2.53 3.8
-
 2-dehydro-3-deoxy-L-lyxonate pH 8.0, 25°C Escherichia coli
4.1.2.53 5.1
-
 2-dehydro-3-deoxy-L-rhamnonate pH 8.0, 25°C Escherichia coli