Literature summary for 4.2.1.90 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.

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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
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

Crystallization (Comment)

Organism

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

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

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

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.06	-	L-mannonate	pH 7.9, 25°C, wild-type enzyme	Salmonella enterica subsp. enterica serovar Typhimurium
0.15	-	L-Rhamnonate	pH 7.9, 25°C, wild-type enzyme	Escherichia coli K-12
0.15	-	L-mannonate	pH 7.9, 25°C, wild-type enzyme	Escherichia coli K-12
0.25	-	L-Rhamnonate	pH 7.9, 25°C, wild-type enzyme	Salmonella enterica subsp. enterica serovar Typhimurium
1.6	-	L-lyxonate	pH 7.9, 25°C, wild-type enzyme	Salmonella enterica subsp. enterica serovar Typhimurium
2	-	L-lyxonate	pH 7.9, 25°C, wild-type enzyme	Escherichia coli K-12

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Escherichia coli K-12
Mg2+	-	Salmonella enterica subsp. enterica serovar Typhimurium

Molecular Weight [Da]

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

Organism

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

Reaction

Reaction	Comment	Organism	Reaction ID
L-rhamnonate = 2-dehydro-3-deoxy-L-rhamnonate + H2O	reaction mechanism of the RhamD-catalyzed reaction, overview	Escherichia coli K-12	a
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-gulonate	low activity	Escherichia coli K-12	? + H2O	-	?
L-lyxonate	-	Escherichia coli K-12	? + H2O	-	?
L-lyxonate	-	Salmonella enterica subsp. enterica serovar Typhimurium	? + H2O	-	?
L-mannonate	-	Escherichia coli K-12	? + H2O	-	?
L-mannonate	-	Salmonella enterica subsp. enterica serovar Typhimurium	? + H2O	-	?
L-rhamnonate	i.e. 6-deoxy-L-mannonate	Escherichia coli K-12	2-dehydro-3-deoxy-L-rhamnonate + H2O	-	?
L-rhamnonate	i.e. 6-deoxy-L-mannonate	Salmonella enterica subsp. enterica serovar Typhimurium	2-dehydro-3-deoxy-L-rhamnonate + H2O	-	?
additional information	stereochemical course of the RhamD-catalyzed reaction, overview	Escherichia coli K-12	?	-	?

Subunits

Subunits	Comment	Organism
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
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

Synonyms	Comment	Organism
More	the enzyme belongs to the enolase superfamily of enzymes	Escherichia coli K-12
More	the enzyme belongs to the enolase superfamily of enzymes	Salmonella enterica subsp. enterica serovar Typhimurium
RhamD	-	Escherichia coli K-12
RhamD	-	Salmonella enterica subsp. enterica serovar Typhimurium
YfaW	-	Escherichia coli K-12

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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