Literature summary for 2.1.1.235 extracted from

Chen, H.; Yamase, H.; Murakami, K.; Chang, C.W.; Zhao, L.; Zhao, Z.; Liu, H.W.
Expression, purification, and characterization of two N,N-dimethyltransferases, tylM1 and desVI, involved in the biosynthesis of mycaminose and desosamine (2002), Biochemistry, 41, 9165-9183.

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Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli. Heterologous expression of the tylM1 gene in the desVI deletion mutant of Streptomyces venezuelae. TylM1 is a competent substitute for DesVI	Streptomyces fradiae

General Stability

General Stability	Organism
concentrated TylM1 protein is stable and can endure repeated freeze/thaw cycles without losing noticeable activity	Streptomyces fradiae
enzyme	Streptomyces fradiae
inclusion of 0.1 mM S-adenosyl-L-methioninet in buffers is crucial to prevent TylM1 from precipitating during the purification	Streptomyces fradiae
TylM1 is unstable at low concentrations, requiring the addition of bovine serum albumin (1 mg/mL) to the assay buffer during kinetic studies of the	Streptomyces fradiae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.0468	-	dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae
0.0594	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae
0.0989	-	S-adenosyl-L-methionine	pH 7.5, 24°C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae
0.117	-	S-adenosyl-L-methionine	pH 7.5, 24°C, cosubstrate: dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae
0.1184	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae

Metals/Ions

Metals/Ions	Comment	Organism	Structure
additional information	TylM1 is not a Mg2+-dependent methyltransferase	Streptomyces fradiae

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
27000	-	2 * 27000, SDS-PAGE	Streptomyces fradiae
27427	-	2 * 27427, calculated from sequence	Streptomyces fradiae
55200	-	gel filtration	Streptomyces fradiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae	the enzyme is involved in the biosynthesis of mycaminose, an essential structural component of the macrolide antibiotic tylosin poduced by Streptomyces fradiae	2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces fradiae	P95748	-	-

Purification (Commentary)

Purification (Comment)	Organism
inclusion of 0.1 mM AdoMet in buffers is crucial to prevent TylM1 from precipitating during the purification	Streptomyces fradiae

Storage Stability

Storage Stability	Organism
-80 °C concentrated TylM1 protein is stable for at least 2 years	Streptomyces fradiae

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2 S-adenosyl-L-methionine + dTDP-3-amino-3,4,6-trideoxy-alpha-xylo-hexopyranose	kcat/KM is about 3fold lower than kcat/Km for the natural substrated TDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae	2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,4,6-trideoxy-alpha-xylo-hexopyranose	i.e. TDP-alpha-D-desosamine	?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	the enzyme is involved in the biosynthesis of mycaminose, an essential structural component of the macrolide antibiotic tylosin poduced by Streptomyces fradiae	Streptomyces fradiae	2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose	-	?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	TylM1 has relaxed specificity toward its sugar substrate. The kcat/KM for dTDP-3-amino-4,6-dideoxy-alpha-D-glucopyranose is about 3fold lower than kcat/Km for the natural substrated TDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae	2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose	-	?
S-adenosyl-L-methionine + dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose	chemically prepared monomethylated compound is a substrate for TylM1 and could be swiftly converted by TylM1 to the dimethylated product. TylM1 catalyzes an N,N-dimethylation reaction by way of a monomethylated intermediate. Since the monomethylated amino group is intrinsically a better nucleophile than the unsubstituted amino group, the reaction rate of this SN2-type methyl transfer reaction is expected to be higher for the second half reaction than for the first methylation reaction	Streptomyces fradiae	S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 27000, SDS-PAGE	Streptomyces fradiae
homodimer	2 * 27427, calculated from sequence	Streptomyces fradiae

Synonyms

Synonyms	Comment	Organism
TylM1	-	Streptomyces fradiae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
24	-	assay at	Streptomyces fradiae

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.09	-	S-adenosyl-L-methionine	pH 7.5, 24°C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae
0.12	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae
0.165	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae
0.54	-	dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae
0.86	-	S-adenosyl-L-methionine	pH 7.5, 24°C, cosubstrate: 0.89 mM dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae

pH Optimum

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

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	-	Streptomyces fradiae

General Information

General Information	Comment	Organism
physiological function	the enzyme catalyzes the N,N-dimethylation step in the biosynthesis of mycaminose. Mycaminose is an aminohexose found in several macrolide antibiotics. The sugar contains a C-3 N,N-dimethylamino group which confers the biological activity of these unusual sugar	Streptomyces fradiae

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
0.91	-	S-adenosyl-L-methionine	pH 7.5, 24°C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae
1	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae
2.78	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae
7.35	-	S-adenosyl-L-methionine	pH 7.5, 24°C, cosubstrate: dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae
11.5	-	dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose	pH 7.5, 24°C	Streptomyces fradiae

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Release 2023.1 (January 2023)