Literature summary for 6.3.2.4 extracted from

Batson, S.; de Chiara, C.; Majce, V.; Lloyd, A.J.; Gobec, S.; Rea, D.; Fueloep, V.; Thoroughgood, C.W.; Simmons, K.J.; Dowson, C.G.; Fishwick, C.W.G.; de Carvalho, L.P.S.; Roper, D.I.
Inhibition of D-Ala D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine (2017), Nat. Commun., 8, 1939 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene ddlB, recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(lambdaDE3)StarpRosetta	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His-tagged enzyme in complex with ATP and D-alanyl-D-alanine, or with ATD and D-alanyl-D-alanine, or with inhibitor D-cycloserine and ADP, hanging drop method, mixing of 0.002 ml of 12 mg/ml protein in 50 mM HEPES, pH 7.5, 150 mM KCl, 0.5 mM EDTA, 5 mM ATP or 5 mM ADP, and 50 mM D-alanyl-D-alanine, with 0.002 ml of reservoir solution containing 200 mM MgCl2, 25% PEG 3350, 100 mM TrisHCl pH 8.0, 18°C, X-ray diffraction structure determination and analysis at 1.4-1.65 A resolution, active site region of EcDdlB in the different complexes, overview	Escherichia coli

Crystallization (Comment)

Organism

purified recombinant His-tagged enzyme in complex with ATP and D-alanyl-D-alanine, or with ATD and D-alanyl-D-alanine, or with inhibitor D-cycloserine and ADP, hanging drop method, mixing of 0.002 ml of 12 mg/ml protein in 50 mM HEPES, pH 7.5, 150 mM KCl, 0.5 mM EDTA, 5 mM ATP or 5 mM ADP, and 50 mM D-alanyl-D-alanine, with 0.002 ml of reservoir solution containing 200 mM MgCl2, 25% PEG 3350, 100 mM TrisHCl pH 8.0, 18°C, X-ray diffraction structure determination and analysis at 1.4-1.65 A resolution, active site region of EcDdlB in the different complexes, overview

Escherichia coli

Inhibitors

Inhibitors	Comment	Organism	Structure
phospho-D-cycloserine	DCSP, inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine, DCS, bimodal mechanism of action of the inhibitor. The compound also inhibits the phosphatase activity of EcDdlB, formation of phospho-D-cycloserine by EcDdlB in solution a positional isotope exchange (PIX) reaction, a phosphatase activity of EcDdlB is detected as a side-reaction alongside the positional isotope exchange (PIX) reaction. DCS is the specific acceptor of the ATP gamma-P group during the phosphate transfer reaction by the enzyme, and DCS inhibits EcDdlB phosphatase activity	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	steady-state kinetics of ATP hydrolysis	Escherichia coli

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + 2 D-alanine	Escherichia coli	-	ADP + phosphate + D-alanyl-D-alanine	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P07862	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(lambdaDE3)StarpRosetta by nickel affinity chromatography and gel filtration	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + 2 D-alanine	-	Escherichia coli	ADP + phosphate + D-alanyl-D-alanine	-	?

Synonyms

Synonyms	Comment	Organism
D-Ala:D-Ala ligase	-	Escherichia coli
EcDdlB	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Escherichia coli

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
additional information	-	additional information	inhibiton kinetics	Escherichia coli