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Information on EC 2.1.1.235 - dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose N,N-dimethyltransferase and Organism(s) Streptomyces fradiae and UniProt Accession P95748
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2.1.1.235 dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose N,N-dimethyltransferaseIUBMB Comments
The enzyme is involved in the biosynthesis of mycaminose, an essential structural component of the macrolide antibiotic tylosin, which is produced by the bacterium Streptomyces fradiae.
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The taxonomic range for the selected organisms is: Streptomyces fradiae
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Synonyms
tylm1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose 3-N,N-dimethyltransferase
The enzyme is involved in the biosynthesis of mycaminose, an essential structural component of the macrolide antibiotic tylosin, which is produced by the bacterium Streptomyces fradiae.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 S-adenosyl-L-methionine + dTDP-3-amino-3,4,6-trideoxy-alpha-xylo-hexopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-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
i.e. TDP-alpha-D-desosamine
-
?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
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
S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-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
-
-
?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
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
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
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
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
the enzyme is involved in biosynthesis of D-mycaminose. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar
-
-
?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-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
-
-
?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-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
-
-
?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
i.e dTDP-mycaminose, binding pocket structure and binding mechanism, overview
analysis of the binding structure of the dTDP-sugar in enzyme TylM1 active site, overview. Only a water molecule is expelled from the active site to accommodate one of the methyl substituents on the C-3' amino group
-
?
additional information
?
-
the enzyme also catalyzes the methylation of dTDP-3-amino-3,6-dideoxy-alpha-D-galactopyranose, EC 2.1.1.236
-
-
?
additional information
?
-
-
the enzyme also catalyzes the methylation of dTDP-3-amino-3,6-dideoxy-alpha-D-galactopyranose, EC 2.1.1.236
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
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
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
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
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
the enzyme is involved in biosynthesis of D-mycaminose. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar
-
-
?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-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
-
-
?
additional information
?
-
the enzyme also catalyzes the methylation of dTDP-3-amino-3,6-dideoxy-alpha-D-galactopyranose, EC 2.1.1.236
-
-
?
additional information
?
-
-
the enzyme also catalyzes the methylation of dTDP-3-amino-3,6-dideoxy-alpha-D-galactopyranose, EC 2.1.1.236
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
TylM1 is not a Mg2+-dependent methyltransferase
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0468
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24°C
0.045
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, wild-type enzyme
0.0594
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24°C
0.079
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH and temperature not specified in the publication
0.1184
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24°C
0.67
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, mutant enzyme H123A
0.93
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, mutant enzyme H123N
0.0989
S-adenosyl-L-methionine
pH 7.5, 24°C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
0.117
S-adenosyl-L-methionine
pH 7.5, 24°C, cosubstrate: dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
additional information
additional information
kinetic analysis of wild-type enzyme and mutants
-
additional information
additional information
-
kinetic analysis of wild-type enzyme and mutants
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.54
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24°C
0.0126
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, mutant enzyme H123N
0.046
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, mutant enzyme H123A
0.165
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24°C
0.172
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, wild-type enzyme
0.75
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH and temperature not specified in the publication
0.09
S-adenosyl-L-methionine
pH 7.5, 24°C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
0.86
S-adenosyl-L-methionine
pH 7.5, 24°C, cosubstrate: 0.89 mM dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
11.5
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH 7.5, 24°C
0.0135
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, mutant enzyme H123N
0.069
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, mutant enzyme H123A
3.8
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
pH 8.5, 37°C, wild-type enzyme
0.91
S-adenosyl-L-methionine
pH 7.5, 24°C, cosubstrate: 1.28 mM dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
7.35
S-adenosyl-L-methionine
pH 7.5, 24°C, cosubstrate: dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
additional information
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
physiological function
the enzyme is involved in biosynthesis of D-mycaminose. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar
physiological function
enzyme TylM1 is a dimethyltransferase from Streptomyces fradiae involved in the production of dTDP-mycaminose
physiological function
CH-O hydrogen bonding play roles in modulating the catalytic efficiency of TylM1
additional information
structure model of TylM1 with bound S-adenosyl-L-methionine and dTDP-phenol: Model of the Michaelis complex in stereo. The C-3' amino group is positioned to attack the methyl group of S-adenoyl-L-methionine. dTDP-mycaminose binding pocket structure, overview
additional information
-
structure model of TylM1 with bound S-adenosyl-L-methionine and dTDP-phenol: Model of the Michaelis complex in stereo. The C-3' amino group is positioned to attack the methyl group of S-adenoyl-L-methionine. dTDP-mycaminose binding pocket structure, overview
additional information
active site structures of WT TylM1 and the Tyr14 and Ser120 mutants, structure modeling, overview. Quantum mechanical calculations of the activation barrier energies of wild-type TylM1 and the Tyr14 mutants suggest that substitutions which abrogate hydrogen bonding with the AdoMet methyl group impair methyl transfer
additional information
-
active site structures of WT TylM1 and the Tyr14 and Ser120 mutants, structure modeling, overview. Quantum mechanical calculations of the activation barrier energies of wild-type TylM1 and the Tyr14 mutants suggest that substitutions which abrogate hydrogen bonding with the AdoMet methyl group impair methyl transfer
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme in complex with S-adenosyl-L-homocysteine and dTDP-3-N-methylamino-3,6-dideoxyglucose., X-ray diffraction structure determination and analysis at 1.6 A resolution
hanging drop method of vapor diffusion method, high resolution X-ray structures of TylM1, one in which the enzyme contains bound SAM and dTDP-phenol and the second in which the protein is complexed with S-adenosyl-L-homocysteine and dTDP-3-amino-3,6-dideoxyglucose, its natural substrate. Combined, these two structures, solved to 1.35 A and 1.79 A resolution, respectively, show the orientations of SAM and the dTDP-linked sugar substrate within the active site region. Specifically, the C-30 amino group of the hexose is in the correct position for an in-line attack at the reactive methyl group of S-adenosyl-L-methionine. High-resolution X-ray models show that the observed perturbations in the kinetic constants of the mutant enzynes H123A and H123N are not due to major changes in their threedimensional folds. Most likely the proton on the C-3' amino group is transferred to one of the water molecules lining the active site pocket as catalysis proceeds
purified enzyme TylM1 mutants Y14F, Y14pAF, and S120A in complex with SAH and dTDP-phenol, X-ray diffraction structure determination and analysis at 1.37-1.78 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H123A
mutant protein retains catalytic activity, the kcat/Km is reduced to 1.8% compared to the wild-type enzyme
H123N
mutant protein retains catalytic activity, the kcat/Km is reduced to 0.37% compared to the wild-type enzyme
S120A
site-directed mutagenesis, the mutant exhibits a modest decrease in its catalytic efficiency compared to wild-type
Y14F
site-directed mutagenesis, the mutation results in an approximately 30fold decrease in catalytic efficiency compared to wild-type
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
concentrated TylM1 protein is stable and can endure repeated freeze/thaw cycles without losing noticeable activity
inclusion of 0.1 mM S-adenosyl-L-methioninet in buffers is crucial to prevent TylM1 from precipitating during the purification
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
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
inclusion of 0.1 mM AdoMet in buffers is crucial to prevent TylM1 from precipitating during the purification
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
the enzyme is involved in biosynthesis of D-mycaminose. D-Mycaminose is an unusual dideoxy sugar found attached to the antibiotic tylosin, a commonly used veterinarian therapeutic
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
Biochemistry
41
9165-9183
2002
Streptomyces fradiae (P95748)
Carney, A.E.; Holden, H.M.
Molecular architecture of TylM1 from Streptomyces fradiae: an N,N-dimethyltransferase involved in the production of dTDP-D-mycaminose
Biochemistry
50
780-787
2011
Streptomyces fradiae (P95748), Streptomyces fradiae
Gandecha, A.R.; Large, S.L.; Cundliffe, E.
Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome
Gene
184
197-203
1997
Streptomyces fradiae (P95748)
Thoden, J.B.; Holden, H.M.
Production of a novel N-monomethylated dideoxysugar
Biochemistry
53
1105-1107
2014
Streptomyces fradiae (P95748), Streptomyces fradiae
Fick, R.J.; Horowitz, S.; McDole, B.G.; Clay, M.C.; Mehl, R.A.; Al-Hashimi, H.M.; Scheiner, S.; Trievel, R.C.
Structural and functional characterization of sulfonium carbon-oxygen hydrogen bonding in the deoxyamino sugar methyltransferase TylM1
Biochemistry
58
2152-2159
2019
Streptomyces fradiae (P95748), Streptomyces fradiae
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