Literature summary for 2.7.1.119 extracted from

Sugimoto, N.; Takakura, Y.; Shiraki, K.; Honda, S.; Takaya, N.; Hoshino, T.; Nakamura, A.
Directed evolution for thermostabilization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus (2013), Biosci. Biotechnol. Biochem., 77, 2234-2241.

Search for more literature for 2.7.1.119

Cloned(Commentary)

Cloned (Comment)	Organism
gene hyg, subcloning in Escherichia coli strain JM109, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3), recombinant enzyme expression from plasmid pT8S-P31 in Thermus thermophilus strain B27 TH104 providing hygromycin B resistance up to 60°C, while recombinant expression of a mutant variant HYG10, containing seven substitutions and a duplication, provides HgB resistance up to 74°C	Streptomyces hygroscopicus

Cloned (Comment)

Organism

gene hyg, subcloning in Escherichia coli strain JM109, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3), recombinant enzyme expression from plasmid pT8S-P31 in Thermus thermophilus strain B27 TH104 providing hygromycin B resistance up to 60°C, while recombinant expression of a mutant variant HYG10, containing seven substitutions and a duplication, provides HgB resistance up to 74°C

Streptomyces hygroscopicus

Protein Variants

Protein Variants	Comment	Organism
L79F/S83L/G189R/E237K/S252P/D285N/L290M	random mutagenesis, mutant HYG10 contains seven substitutions and a duplication, it is thermostabilized compared to the wild-type enzyme, three-dimensional structure analysis, overview. Substitutions G189R, E237K, and D285N are located at the protein surface. The mutant shows altered kinetics at 45°C compared to the wild-type enzyme	Streptomyces hygroscopicus

Inhibitors

Inhibitors	Comment	Organism	Structure
hygromycin B	substrate inhibition	Streptomyces hygroscopicus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.0167	-	hygromycin B	pH 7.5, 45°C, recombinant wild-type enzyme YG	Streptomyces hygroscopicus
0.03	-	hygromycin B	pH 7.5, 45°C, recombinant mutant enzyme HYG10	Streptomyces hygroscopicus
0.0423	-	ATP	pH 7.5, 45°C, recombinant wild-type enzyme YG	Streptomyces hygroscopicus
0.0822	-	ATP	pH 7.5, 45°C, recombinant mutant enzyme HYG10	Streptomyces hygroscopicus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Streptomyces hygroscopicus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
37980	-	-	Streptomyces hygroscopicus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + hygromycin B	Streptomyces hygroscopicus	-	ADP + 7''-O-phosphohygromycin B	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces hygroscopicus	P09979	gene hyg	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme fom Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, and gel filtration	Streptomyces hygroscopicus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + hygromycin B	-	Streptomyces hygroscopicus	ADP + 7''-O-phosphohygromycin B	-	?

Subunits

Subunits	Comment	Organism
?	x * 37980, wild-type enzyme, sequence calculation, x * 38348, enzyme mutant HYG10, sequence calculation	Streptomyces hygroscopicus

Synonyms

Synonyms	Comment	Organism
hygromycin B phosphotransferase	-	Streptomyces hygroscopicus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	45	assay at	Streptomyces hygroscopicus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
35	-	T1/2 of wild-type enzyme HYG	Streptomyces hygroscopicus
48	74	T1/2 of mutant enzyme HYG10, Tmax is 74°C	Streptomyces hygroscopicus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
2.5	-	ATP	pH 7.5, 45°C, recombinant wild-type enzyme YG	Streptomyces hygroscopicus
5.6	-	ATP	pH 7.5, 45°C, recombinant mutant enzyme HYG10	Streptomyces hygroscopicus
6.4	-	hygromycin B	pH 7.5, 45°C, recombinant wild-type enzyme YG	Streptomyces hygroscopicus
8	-	hygromycin B	pH 7.5, 45°C, recombinant mutant enzyme HYG10	Streptomyces hygroscopicus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Streptomyces hygroscopicus

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Streptomyces hygroscopicus

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.61	-	hygromycin B	pH 7.5, 45°C, recombinant wild-type enzyme YG	Streptomyces hygroscopicus
1.27	-	hygromycin B	pH 7.5, 45°C, recombinant mutant enzyme HYG10	Streptomyces hygroscopicus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
59.1	-	ATP	pH 7.5, 45°C, recombinant wild-type enzyme YG	Streptomyces hygroscopicus
68.1	-	ATP	pH 7.5, 45°C, recombinant mutant enzyme HYG10	Streptomyces hygroscopicus
267	-	hygromycin B	pH 7.5, 45°C, recombinant mutant enzyme HYG10	Streptomyces hygroscopicus
383	-	hygromycin B	pH 7.5, 45°C, recombinant wild-type enzyme YG	Streptomyces hygroscopicus