Literature summary for 2.7.1.176 extracted from

Rocker, A.; Peschke, M.; Kittilae, T.; Sakson, R.; Brieke, C.; Meinhart, A.
The ng_zeta1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis (2018), Nat. Commun., 9, 1686 .

Search for more literature for 2.7.1.176

Cloned(Commentary)

Cloned (Comment)	Organism
gene pezT, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)-RIL, Ngzeta_1 expression in Escherichia coli cells causes a lytic phenotype compensated by ngepsilon_1 coexpression. Growth curves of Escherichia coli C41(DE3) cells expressing ngzeta_1 wild-type and ngzeta_1 mutant K115A show that when ngzeta_1 is expressed in Escherichia coli cells, a strong lytic phenotype 30 min post-induction occurs	Neisseria gonorrhoeae

Cloned (Comment)

Organism

gene pezT, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)-RIL, Ngzeta_1 expression in Escherichia coli cells causes a lytic phenotype compensated by ngepsilon_1 coexpression. Growth curves of Escherichia coli C41(DE3) cells expressing ngzeta_1 wild-type and ngzeta_1 mutant K115A show that when ngzeta_1 is expressed in Escherichia coli cells, a strong lytic phenotype 30 min post-induction occurs

Neisseria gonorrhoeae

Crystallization (Commentary)

Crystallization (Comment)	Organism
wild-type ng_epsilon1/ng_zeta1 complex with bound substrate UDP-muramic acid and product UDP-muramic acid 4'-phosphate and of selenomethionine-labeled ng_epsilon1/ng_zeta1_K115A complex mutant, X-ray diffraction structure determination and analysis at 2.4-2.8 A resolution	Neisseria gonorrhoeae

Protein Variants

Protein Variants	Comment	Organism
K115A	site-directed mutagenesis, inactive mutant	Neisseria gonorrhoeae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics	Neisseria gonorrhoeae
0.23	-	UDP-muramic acid	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
0.31	-	ATP	with UDP-alpha-D-glucose, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
0.34	-	ATP	with UDP-N-acetyl-alpha-D-glucosamine, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
2.6	-	ATP	with UDP-muramic acid, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
2.8	-	UDP-N-acetyl-alpha-D-glucosamine	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
6.4	-	UDP-alpha-D-glucose	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Neisseria gonorrhoeae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + UDP-N-acetyl-alpha-D-glucosamine	Neisseria gonorrhoeae	-	ADP + UDP-N-acetyl-alpha-D-glucosamine 3'-phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Neisseria gonorrhoeae	A0A5K1QBJ4	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)-RIL by nickel affinity chromatography and gel filtration	Neisseria gonorrhoeae

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + UDP-alpha-D-glucose	-	Neisseria gonorrhoeae	ADP + UDP-alpha-D-glucose 3'-phosphate	-	?
ATP + UDP-muramic acid	ngzeta_1 phosphorylates UDP-muramic acid (UNAM) at the 4'-hydroxy group, binding structure analysis of substrate and product. The phosphate group on UNAM is attached to the C4'-OH and not to the C3'-OH group atom, different to what has been described for the hitherto characterized zeta UNAG kinases	Neisseria gonorrhoeae	ADP + UDP-muramic acid 4'-phosphate	-	?
ATP + UDP-N-acetyl-alpha-D-glucosamine	-	Neisseria gonorrhoeae	ADP + UDP-N-acetyl-alpha-D-glucosamine 3'-phosphate	-	?
additional information	UDP-sugar specificity of the recombinant enzyme, overview. Enzyme ng_zeta1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. The phosphorylation site of Neisseria zeta-toxin is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. Arg181 and Arg175 form a positively charged patch in the active site counteracting the negatively charged phosphate groups	Neisseria gonorrhoeae	?	-	-

Subunits

Subunits	Comment	Organism
More	the enzyme is produced and analyzed in a ng_epsilon1/ng_zeta1 complex	Neisseria gonorrhoeae

Synonyms

Synonyms	Comment	Organism
epsilon/zeta toxin-antitoxin homologue	-	Neisseria gonorrhoeae
Ngzeta_1	-	Neisseria gonorrhoeae
ng_epsilon1 / ng_zeta1	-	Neisseria gonorrhoeae
ng_zeta1 toxin	-	Neisseria gonorrhoeae
PezT	-	Neisseria gonorrhoeae
TA homologue	-	Neisseria gonorrhoeae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Neisseria gonorrhoeae

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.51	-	ATP	with UDP-alpha-D-glucose, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
0.71	-	UDP-alpha-D-glucose	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
1.72	-	UDP-N-acetyl-alpha-D-glucosamine	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
1.76	-	ATP	with UDP-N-acetyl-alpha-D-glucosamine, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
200	-	UDP-muramic acid	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
280	-	ATP	with UDP-muramic acid, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Neisseria gonorrhoeae

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Neisseria gonorrhoeae

General Information

General Information	Comment	Organism
evolution	ngzeta_1 forms a new subclass of zeta-like toxins. Apart from being encoded on a bicistronic operon and harbouring a P-loop motive, a hallmark for ATP/GTP binding proteins, ngzeta_1 is remarkably different from the hitherto functionally characterized streptococcal zeta toxins in its primary sequence. Especially striking is that the P-loop motive is located much closer to the Cterminus when compared with streptococcal zeta toxins. Also the ngepsilon_1 antitoxin has no similarities to any known epsilon protein	Neisseria gonorrhoeae
metabolism	ngzeta_1 drains precursors from peptidoglycan synthesis at multiple stages. Under normal conditions cytosolic levels of UNAM regulate peptidoglycan synthesis by a negative feedback loop inhibiting MurA. In contrast, once ngzeta_1 becomes active, MurA, MurB, and MurC are depleted from their substrates. All phosphorylated precursors are dead-end metabolites, but none of them seems to directly inhibit any enzyme of early peptidoglycan synthesis	Neisseria gonorrhoeae
additional information	the enzyme is produced and analyzed in a ng_epsilon1/ng_zeta1 complex	Neisseria gonorrhoeae
physiological function	zeta-toxins interfere with cell wall synthesis. The phosphorylation site of Neisseria zeta-toxin is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence	Neisseria gonorrhoeae

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.11	-	UDP-alpha-D-glucose	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
0.61	-	UDP-N-acetyl-alpha-D-glucosamine	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
1.65	-	ATP	with UDP-alpha-D-glucose, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
5.18	-	ATP	with UDP-N-acetyl-alpha-D-glucosamine, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
107.7	-	ATP	with UDP-muramic acid, pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae
869.6	-	UDP-muramic acid	pH 6.0, 25°C, recombinant wild-type enzyme ngzeta_1	Neisseria gonorrhoeae