BRENDA - Enzyme Database show
show all sequences of 2.1.1.235

Expression, purification, and characterization of two N,N-dimethyltransferases, tylM1 and desVI, involved in the biosynthesis of mycaminose and desosamine

Chen, H.; Yamase, H.; Murakami, K.; Chang, C.W.; Zhao, L.; Zhao, Z.; Liu, H.W.; Biochemistry 41, 9165-9183 (2002)

Data extracted from this reference:

Cloned(Commentary)
Commentary
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
Commentary
Organism
Structure
0.0468
-
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.0594
-
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.0989
-
S-adenosyl-L-methionine
pH 7.5, 24C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
Streptomyces fradiae
0.117
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
additional information
TylM1 is not a Mg2+-dependent methyltransferase
Streptomyces fradiae
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
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
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
Primary Accession No. (UniProt)
Commentary
Textmining
Streptomyces fradiae
P95748
-
-
Purification (Commentary)
Commentary
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
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
714149
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
714149
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
714149
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
714149
Streptomyces fradiae
S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
-
-
-
?
Subunits
Subunits
Commentary
Organism
homodimer
2 * 27000, SDS-PAGE; 2 * 27427, calculated from sequence
Streptomyces fradiae
Temperature Optimum [C]
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
24
-
assay at
Streptomyces fradiae
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.09
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
0.165
-
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.54
-
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.86
-
S-adenosyl-L-methionine
pH 7.5, 24C, cosubstrate: 0.89 mM dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
Streptomyces fradiae
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
assay at
Streptomyces fradiae
Cofactor
Cofactor
Commentary
Organism
Structure
S-adenosyl-L-methionine
-
Streptomyces fradiae
Cloned(Commentary) (protein specific)
Commentary
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
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
S-adenosyl-L-methionine
-
Streptomyces fradiae
General Stability (protein specific)
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] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0468
-
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.0594
-
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.0989
-
S-adenosyl-L-methionine
pH 7.5, 24C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
Streptomyces fradiae
0.117
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
additional information
TylM1 is not a Mg2+-dependent methyltransferase
Streptomyces fradiae
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
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) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
inclusion of 0.1 mM AdoMet in buffers is crucial to prevent TylM1 from precipitating during the purification
Streptomyces fradiae
Storage Stability (protein specific)
Storage Stability
Organism
-80 C concentrated TylM1 protein is stable for at least 2 years
Streptomyces fradiae
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
714149
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
714149
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
714149
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
714149
Streptomyces fradiae
S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
homodimer
2 * 27000, SDS-PAGE; 2 * 27427, calculated from sequence
Streptomyces fradiae
Temperature Optimum [C] (protein specific)
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
24
-
assay at
Streptomyces fradiae
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.09
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
0.165
-
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.54
-
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
0.86
-
S-adenosyl-L-methionine
pH 7.5, 24C, cosubstrate: 0.89 mM dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
Streptomyces fradiae
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
assay at
Streptomyces fradiae
General Information
General Information
Commentary
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
General Information (protein specific)
General Information
Commentary
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
Commentary
Organism
Structure
0.91
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
2.78
-
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
7.35
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.91
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
2.78
-
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24C
Streptomyces fradiae
7.35
-
S-adenosyl-L-methionine
pH 7.5, 24C, 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, 24C
Streptomyces fradiae
Other publictions for EC 2.1.1.235
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [C]
Temperature Range [C]
Temperature Stability [C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [C] (protein specific)
Temperature Range [C] (protein specific)
Temperature Stability [C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
735676
Thoden
Production of a novel N-monome ...
Streptomyces fradiae
Biochemistry
53
1105-1107
2014
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1
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714271
Carney
Molecular architecture of TylM ...
Streptomyces fradiae
Biochemistry
50
780-787
2011
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2
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3
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714149
Chen
Expression, purification, and ...
Streptomyces fradiae
Biochemistry
41
9165-9183
2002
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1
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4
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5
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1
3
1
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715103
Gandecha
Analysis of four tylosin biosy ...
Streptomyces fradiae
Gene
184
197-203
1997
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