Literature summary for 6.3.2.9 extracted from

Sink, R.; Kotnik, M.; Zega, A.; Barreteau, H.; Gobec, S.; Blanot, D.; Dessen, A.; Contreras-Martel, C.
Crystallographic study of peptidoglycan biosynthesis enzyme MurD: domain movement revisited (2016), PLoS ONE, 11, e0152075 .

Application

Application	Comment	Organism
drug development	the enzyme is a target in the development of potential antibiotics designed to target the peptidoglycan biosynthetic pathway	Escherichia coli

Cloned(Commentary)

Cloned (Comment)	Organism
gene murD, recombinant expression of His6-tagged enzyme in Escherichia coli strain DH5alpha	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified free MurD and MurD complexed with the tetrahedral reaction intermediate, hanging drop vapor diffusion method, for free enzyme crystals: mixing of 0.002 ml of 3 mg/ml protein in 20 mM HEPES, pH 5.6, 1 mM DTT, and 1 mM NaN3, with 0.002 ml of reservoir solution containing 1.8 M (NH4)2SO4, 7% v/v 2-methyl-2,4-pentanediol, and 0.1 MMES, pH 5.6, 15°C, 48 h, for complexed enzyme crystals: mixing of 0.002 ml of 4 mg/ml protein in 20 mM HEPES, pH 7.4, 1 mM DTT, 1 mM NaN3, 5 mMAMP-PNP, and 1 mM UDP-N-acetyl-alpha-D-muramoyl-L-alanine, with 0.002 ml of reservoir solution containing 1.8 M Na-malonate, pH 7.0, 15°C, 48 h, X-ray diffraction structure determination and analysis at 1.84-1.90 A resolution	Escherichia coli

Crystallization (Comment)

Organism

purified free MurD and MurD complexed with the tetrahedral reaction intermediate, hanging drop vapor diffusion method, for free enzyme crystals: mixing of 0.002 ml of 3 mg/ml protein in 20 mM HEPES, pH 5.6, 1 mM DTT, and 1 mM NaN3, with 0.002 ml of reservoir solution containing 1.8 M (NH4)2SO4, 7% v/v 2-methyl-2,4-pentanediol, and 0.1 MMES, pH 5.6, 15°C, 48 h, for complexed enzyme crystals: mixing of 0.002 ml of 4 mg/ml protein in 20 mM HEPES, pH 7.4, 1 mM DTT, 1 mM NaN3, 5 mMAMP-PNP, and 1 mM UDP-N-acetyl-alpha-D-muramoyl-L-alanine, with 0.002 ml of reservoir solution containing 1.8 M Na-malonate, pH 7.0, 15°C, 48 h, X-ray diffraction structure determination and analysis at 1.84-1.90 A resolution

Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	ordered kinetic mechanism	Escherichia coli

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytoplasm	-	Escherichia coli	5737	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P14900	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme from Escherichia coli strain DH5alpha by nickel affinity chromatography and dialysis	Escherichia coli

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + UDP-N-acetyl-alpha-D-muramoyl-L-alanine + D-glutamate = ADP + phosphate + UDP-N-acetyl-alpha-D-muramoyl-L-alanyl-D-glutamate	reaction via tetrahedral intermediate. The enzyme performs binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. Reaction mechanism and structure analysis, overview	Escherichia coli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + UDP-N-acetyl-alpha-D-muramoyl-L-alanine + D-glutamate	-	Escherichia coli	ADP + phosphate + UDP-N-acetyl-alpha-D-muramoyl-L-alanyl-D-glutamate	-	?
additional information	enzyme MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
MurD	-	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	ATP binding to the enzyme causes conformational changes, overview	Escherichia coli

General Information

General Information	Comment	Organism
metabolism	the enzyme is involved in the peptidoglycan biosynthesis	Escherichia coli
additional information	conformational changes of MurD occur upon ligand binding. Upon binding of ATP and UDP-N-acetyl-alpha-D-muramoyl-L-alanine, there is a closing rotation of the C-terminal domain, which does not occur before ATP is bound, targeted molecular dynamics simulation	Escherichia coli
physiological function	MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala, generating the dipeptide moiety L-Ala-D-Glu in peptidoglycan biosynthesis	Escherichia coli