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Information on EC 2.2.1.9 - 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase and Organism(s) Escherichia coli and UniProt Accession P17109

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EC Tree
IUBMB Comments
Requires Mg2+ for maximal activity. This enzyme is involved in the biosynthesis of vitamin K2 (menaquinone). In most anaerobes and all Gram-positive aerobes, menaquinone is the sole electron transporter in the respiratory chain and is essential for their survival. It had previously been thought that the products of the reaction were (1R,6R)-6-hydroxy-2-succinylcyclohexa-2,4-diene-1-carboxylate (SHCHC), pyruvate and CO2 but it is now known that two separate enzymes are involved: this enzyme and EC 4.2.99.20, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase. Under basic conditions, the product can spontaneously lose pyruvate to form SHCHC.
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Escherichia coli
UNIPROT: P17109
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
shchc synthase, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, ecmend, sephchc synthase, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate synthase
-
2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase
-
SEPHCHC synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
isochorismate + 2-oxoglutarate = 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
isochorismate:2-oxoglutarate 4-oxopentanoatetransferase (decarboxylating)
Requires Mg2+ for maximal activity. This enzyme is involved in the biosynthesis of vitamin K2 (menaquinone). In most anaerobes and all Gram-positive aerobes, menaquinone is the sole electron transporter in the respiratory chain and is essential for their survival. It had previously been thought that the products of the reaction were (1R,6R)-6-hydroxy-2-succinylcyclohexa-2,4-diene-1-carboxylate (SHCHC), pyruvate and CO2 but it is now known that two separate enzymes are involved: this enzyme and EC 4.2.99.20, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase. Under basic conditions, the product can spontaneously lose pyruvate to form SHCHC.
CAS REGISTRY NUMBER
COMMENTARY hide
122007-88-9
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(+)-5-(carboxymethoxy)-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid + 2-oxoglutarate
? + CO2
show the reaction diagram
-
analogue of isochorismate, weaker binding to enzyme. Only the (+)-enantiomer is a substrate
-
-
?
(5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate + 2-oxoglutarate
(1R,2S,5S,6S)-2-succinyl-5,6-dihydroxycyclohex-3-ene-1-carboxylate + CO2
show the reaction diagram
-
-
-
?
(5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate + 2-oxoglutarate
2-succinyl-6-amino-5-hydroxycyclohex-3-ene-1-carboxylate + CO2
show the reaction diagram
-
-
-
?
isochorismate + 2-oxoglutarate
5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2
show the reaction diagram
isochorismate + 2-oxoglutarate
5-enolpyruvoyl-6-hydroxy-2-succinylcyclohex-3-ene-1-carboxylate + CO2
show the reaction diagram
additional information
?
-
-
MenD participates in the menaquinone (vitamin K2) biosynthetic pathway
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
isochorismate + 2-oxoglutarate
5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2
show the reaction diagram
isochorismate + 2-oxoglutarate
5-enolpyruvoyl-6-hydroxy-2-succinylcyclohex-3-ene-1-carboxylate + CO2
show the reaction diagram
-
-
-
?
additional information
?
-
-
MenD participates in the menaquinone (vitamin K2) biosynthetic pathway
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thiamine diphosphate
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-[hydroxy(methoxy)phosphoryl]-4-oxobutanoic acid
-
competitive with respect to 2-oxoglutarate, uncompetitive with respect to (+)-5-(carboxymethoxy)-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid. Compound is proposed to bind to the active site and react with thiamine diphosphate but not proceed forward to products by cleavage of the C-P bond
methyl succinylphosphonate
-
dead-end inhibitor
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thiamine diphosphate
-
required, Km: 0.0024 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0042 - 0.109
(+)-5-(carboxymethoxy)-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid
0.0026 - 1.5
2-oxoglutarate
0.00008 - 0.053
isochorismate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12 - 0.4
(+)-5-(carboxymethoxy)-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid
0.0027 - 0.3
2-oxoglutarate
0.0025 - 0.337
isochorismate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1 - 1417
(+)-5-(carboxymethoxy)-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid
0.016 - 98.3
2-oxoglutarate
0.3 - 400
isochorismate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00013
4-[hydroxy(methoxy)phosphoryl]-4-oxobutanoic acid
-
pH 7.4, temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8
-
phosphate buffer
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
the enzyme Men D is not significantly different from other ThDP-dependent enzymes in active-site architecture, cofactor binding, or overall three-dimensional structure, suggesting the use of the canonical enamine intermediate in its catalysis
metabolism
physiological function
-
enzyme is essential for menaquinone biosynthesis
additional information
in the covalent tetrahedral enamine intermediate, all of the bond lengths and angles of its planar thiazolium ring are comparable to those of a similar enzyme-free thiamine diphosphate adduct, complex crystal structure determination and analysis, NMR structure analysis and modeling, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystallization of the apoenzyme and holoenzyme forms of MenD. The apoenzyme crystals are obtained by sitting-drop vapour diffusion with 70% MPD. The crystals are too small to collect diffraction data and a search for better conditions is not successful. Single crystals of the holoenzyme with thiamin diphosphate and Mn2+ as cofactors are obtained by the hanging-drop vapour-diffusion method with 35% ethylene glycol as precipitant. Diffraction data are collected on a cryocooled crystal to a resolution of 2.0 A
-
mutant enzyme R395K bound to thiamine diphosphate, hanging drop vapor diffusion method, using 0.2 M ammonium acetate, 0.06 M magnesium formate, 3% (w/v) polyethylene glycol 3350, and 12% (w/v) polyethylene glycol 10000 in 0.1 M Tris-HCl (pH 7.5). Mutant enzyme R395A bound to thiamine diphosphate, hanging drop vapor diffusion method, using 30% (v/v) Jeffamine M-600 (pH 7.0), 11.2% (w/v) polyethylene glycol 3350 and 0.16 M magnesium formate in 0.1 M HEPES (pH 7.5). Mutant enzyme R413A bound to thiamine diphosphate, hanging drop vapor diffusion method, using 0.16 M magnesium formate, 1% (w/v) tacsimate (pH 7.0), 14% (w/v) polyethylene glycol 3350, and 2% (w/v) polyethylene glycol MME 5000 in 0.02 M HEPES (pH 7.0)
purified recombinant enzyme complexed with its tetrahedral reaction intermediate, X-ray diffraction structure determination and analysis
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E55D
-
about 30% decrease in catalytic efficiency
I148L
-
strong decrease in catalytic efficiency
K292Q
-
150% increase in catalytic efficiency
N117R/L478T
the mutant preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate with a more than 70fold higher ratio than that for the wild type enzyme
Q118A
the mutant is completely inactive
R107I
the mutant uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate as substrate with more than 6fold conversion compared to the wild type enzyme
R107K
-
no decrease in catalytic efficiency
R293K
-
slight increase in catalytic efficiency
R395A
the mutant shows a 160 and 1000fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme
R395K
R395K/R413K
the mutant shows a 6300 and 1300fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme
R413A
the mutant is completely inactive
R413K
S32A
-
slight decrease in catalytic efficiency
S391A
-
slight decrease in catalytic efficiency
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE column chromatography and Sephacryl S-200 gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Sieminska, E.A.; Macova, A.; Palmer, D.R.; Sanders, D.A.
Crystallization and preliminary X-ray analysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase (MenD) from Escherichia coli
Acta Crystallogr. Sect. F
61
489-492
2005
Escherichia coli
Manually annotated by BRENDA team
Jiang, M.; Cao, Y.; Guo, Z.F.; Chen, M.; Chen, X.; Guo, Z.
Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity
Biochemistry
46
10979-10989
2007
Escherichia coli
Manually annotated by BRENDA team
Fang, M.; Macova, A.; Hanson, K.L.; Kos, J.; Palmer, D.R.
Using substrate analogues to probe the kinetic mechanism and active site of Escherichia coli MenD
Biochemistry
50
8712-8721
2011
Escherichia coli, Mycobacterium tuberculosis
Manually annotated by BRENDA team
Song, H.; Dong, C.; Qin, M.; Chen, Y.; Sun, Y.; Liu, J.; Chan, W.; Guo, Z.
A thiamine-dependent enzyme utilizes an active tetrahedral intermediate in vitamin K biosynthesis
J. Am. Chem. Soc.
138
7244-7247
2016
Escherichia coli (P17109)
Manually annotated by BRENDA team
Qin, M.; Song, H.; Dai, X.; Chen, Y.; Guo, Z.
Two active site arginines are critical determinants of substrate binding and catalysis in MenD a thiamine-dependent enzyme in menaquinone biosynthesis
Biochem. J.
475
3651-3667
2018
Escherichia coli (P17109)
Manually annotated by BRENDA team
Fries, A.; Mazzaferro, L.S.; Gruening, B.; Bisel, P.; Stibal, K.; Buchholz, P.C.F.; Pleiss, J.; Sprenger, G.A.; Mueller, M.
Alteration of the route to menaquinone towards isochorismate-derived metabolites
ChemBioChem
20
1672-1677
2019
Escherichia coli (P17109)
Manually annotated by BRENDA team