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0.0013 - 0.026
tripolyphosphate
0.01
ATP
pH 8, 37°C, mutant enzyme I303V/I65V/L186V
0.01
ATP
pH 8, 37°C, mutant enzyme I303V/I65V/L186V/N104K
0.026
ATP
G6 mutant, S-adenosylmethionine synthesis
0.045
ATP
RLL and G8 mutants, S-adenosylmethionine synthesis
0.05
ATP
pH 8, 37°C, mutant enzyme I303V/I65V
0.069
ATP
G7 mutants, S-adenosylmethionine synthesis
0.073
ATP
wild-type S-adenosylmethionine synthesis
0.083
ATP
wild-type, pH 8.0
0.087
ATP
G5 mutant S-adenosylmethionine synthesis
0.1
ATP
pH 8, 37°C, mutant enzyme I303V
0.13
ATP
mutant D107C, pH 8.0
0.13
ATP
mutant G105C, pH 8.0
0.17
ATP
pH 8, 37°C, wild-type enzyme
0.18
ATP
mutant G105R1, pH 8.0
0.08
L-methionine
pH 8, 37°C, wild-type enzyme
0.092
L-methionine
wild-type, S-adenosylmethionine synthesis
0.11
L-methionine
wild-type, pH 8.0
0.17
L-methionine
pH 8, 37°C, mutant enzyme I303V/I65V/L186V/N104K
0.18
L-methionine
mutant D107R1, pH 8.0
0.19
L-methionine
mutant D107R1, pH 8.0
0.23
L-methionine
RLL mutant, S-adenosylmethionine synthesis
0.29
L-methionine
mutant D107C, pH 8.0
0.3
L-methionine
G6 mutant, S-adenosylmethionine synthesis
0.3
L-methionine
pH 8, 37°C, mutant enzyme I303V/I65V/L186V
0.42
L-methionine
pH 8, 37°C, mutant enzyme I303V
0.43
L-methionine
pH 8, 37°C, mutant enzyme I303V/I65V
0.45
L-methionine
mutant G105C, pH 8.0
0.49
L-methionine
G7 mutant, S-adenosylmethionine synthesis
0.62
L-methionine
G8 mutant, S-adenosylmethionine synthesis
0.75
L-methionine
mutant G105R1, pH 8.0
0.77
L-methionine
G5 mutant, S-adenosylmethionine synthesis
0.0013
tripolyphosphate
wild type, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine
0.0015
tripolyphosphate
G7 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine
0.0016
tripolyphosphate
G8 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine
0.003
tripolyphosphate
wild type, tripolyphosphatase activity, in absence of S-adenosylmethionine
0.0032
tripolyphosphate
G6 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine
0.0053
tripolyphosphate
G5 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine
0.008
tripolyphosphate
G8 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine
0.011
tripolyphosphate
G6 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine
0.014
tripolyphosphate
RLL mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine
0.015
tripolyphosphate
G7 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine
0.024
tripolyphosphate
G5 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine
0.026
tripolyphosphate
RLL mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine
0.34
ATP
-
37°C, pH 8.0, mutant enzyme I303V
0.43
ATP
-
37°C, pH 8.0, wild-type enzyme
1.72
ATP
-
at pH 8.5 and 45°C
0.03 - 0.038
L-methionine
-
one kinetic form of alpha subunit in the presence of beta subunit
0.065 - 0.08
L-methionine
-
alpha subunit at low L-methionine concentrations
0.08 - 0.09
L-methionine
-
alpha subunit at high L-methionine concentrations. Also one kinetic form of alpha subunit in the presence of beta subunit
0.12
L-methionine
-
37°C, pH 8.0, mutant enzyme I303V
0.18
L-methionine
-
37°C, pH 8.0, wild-type enzyme
0.85
L-methionine
-
at pH 8.5 and 45°C
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0.000000367 - 0.000417
S-adenosylmethionine
0.000000217 - 0.00000167
tripolyphosphate
1.22
ATP
pH 8, 37°C, wild-type enzyme
1.3
ATP
pH 8, 37°C, mutant enzyme I303V
1.94
ATP
pH 8, 37°C, mutant enzyme I303V/I65V
3.28
ATP
pH 8, 37°C, mutant enzyme I303V/I65V/L186V
4.34
ATP
pH 8, 37°C, mutant enzyme I303V/I65V/L186 V/N104K
0.23
L-methionine
mutant D107R, pH 8.0
1.22
L-methionine
pH 8, 37°C, wild-type enzyme
1.3
L-methionine
pH 8, 37°C, mutant enzyme I303V
1.4
L-methionine
mutant G105R, pH 8.0
1.5
L-methionine
wild-type, pH 8.0
1.6
L-methionine
mutant D107C, pH 8.0
1.6
L-methionine
mutant G105C, pH 8.0
1.94
L-methionine
pH 8, 37°C, mutant enzyme I303V/I65V
3.28
L-methionine
pH 8, 37°C, mutant enzyme I303V/I65V/L186V
4.34
L-methionine
pH 8, 37°C, mutant enzyme I303V/I65V/L186V/N104K
0.000000367
S-adenosylmethionine
RLL mutant, S-adenosylmethionine synthesis
0.00000045
S-adenosylmethionine
G6 mutant, S-adenosylmethionine synthesis
0.000000667
S-adenosylmethionine
G8 mutant, S-adenosylmethionine synthesis
0.00000217
S-adenosylmethionine
G5 mutant, S-adenosylmethionine synthesis
0.000012
S-adenosylmethionine
G7 mutant, S-adenosylmethionine synthesis
0.000417
S-adenosylmethionine
wild-type, S-adenosylmethionine synthesis
0.000000217
tripolyphosphate
G8 mutant, tripolyphosphatase activity
0.000000617
tripolyphosphate
G6 mutant, tripolyphosphatase activity
0.000000633
tripolyphosphate
G5 mutant, tripolyphosphatase activity
0.00000122
tripolyphosphate
RLL and G7 mutants, tripolyphosphatase activity
0.00000167
tripolyphosphate
wild-type, tripolyphosphatase activity
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D107C
enzyme activity similar to wild-type, attachment of methanethiosulfonate spin label to form D107R1, increase in Km-value, decrease in kcat value
G105C
enzyme activity similar to wild-type, attachment of methanethiosulfonate spin label to form G105R1, increase in Km-value
I303V/I65V/L186V
product inhibition of the enzyme is reduced via semi-rational modification. The mutant enzyme shows a 42fold increase in Ki(ATP) and a 2.08fold increase in specific activity when compared to wild-type enzyme. Its Ki(ATP) is 0.42 mM and specific acitivity is 3.78 U/mg. Increased Ki(ATP) means reduced product inhibition which enhances accumulation of S-adenosyl-L-methionine. The S-adenosyl-L-methionine produced by the variant could reach to 3.27 mM while S-adenosyl-L-methionine produced by wild-type enzyme is 1.62 mM in the presence of 10 mM substrates
I303V/I65V/L186V/N104K
product inhibition of the enzyme is reduced via semi-rational modification. The mutant enzyme shows a 3.3fold increase in specific activity when compared to wild-type enzyme. Specific acitivity of the mutant enzyme is 6.02 U/mg. Increased Ki(ATP) means reduced product inhibition which enhances accumulation of S-adenosyl-L-methionine. The S-adenosyl-L-methionine produced by the variant could reach to 2.68 mM while S-adenosyl-L-methionine produced by wild-type enzyme is 1.62 mM in the presence of 10 mM substrates
I303V
-
the Km-values for both substrates are slightly less than those of the wild-type enzyme. The variant is successfully produced at a high level (about 800 mg/l) with approximately four-fold higher specific activity than the wild-type enzyme. The recombinant mutant enzyme is covalently immobilized onto the amino resin and epoxy resin in order to obtain a robust biocatalyst to be used in industrial bioreactors. The immobilized preparation using amino resin exhibits the highest activity coupling yield (about 84%), compared with approximately 3% for epoxy resin. The immobilized mutant enzyme is more stable than the soluble enzyme under the reactive conditions, with a half-life of 229.5 h at 37 °C. The Km(ATP) value (0.18 mM) of the immobilized mutant enzyme is about two-fold lower than that of the soluble enzyme. The immobilized enzyme shows high operational stability during 10 consecutive 8 h batches, with the substrate adenosine triphosphate conversion rate above 95% on the 50 mM scale. Compared with the wild-type enzyme, as little as 200 mM sodium p-toluenesulfonate is required to completely overcome the product inhibition by S-adenosyl-L-methionine of I303V mutant enzyme on a 30 mM scale incubation
additional information
mutants D107R1, D105R1, derived from mutants D107C, D105C, by addition of methanethiosulfonate spin label
additional information
recombination of MAT genes from Escherichia coli, Saccharomyces cerevisiae, and Streptomyces spectabilis by DNA shuffling and transformation into Pichia pastoris. In the two best recombinant strains, the MAT activities are respectively 201% and 65% higher than the recombinant strains containing the starting MAT genes, and the SAM concentration increases by 103% and 65%, respectively. A 6.14 g/l of SAM production is reached in a 500 l bioreactor with the best recombinant strain
additional information
-
recombination of MAT genes from Escherichia coli, Saccharomyces cerevisiae, and Streptomyces spectabilis by DNA shuffling and transformation into Pichia pastoris. In the two best recombinant strains, the MAT activities are respectively 201% and 65% higher than the recombinant strains containing the starting MAT genes, and the SAM concentration increases by 103% and 65%, respectively. A 6.14 g/l of SAM production is reached in a 500 l bioreactor with the best recombinant strain
additional information
-
construction of a metK deletion mutant strain MOB1490 from wild-type strain BW25113, complementation by wild-type gene metK, as well as by genes metK from Rickettsia prowazekii and Rickettsia typhi, overview
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Markham, G.D.; Hafner, E.W.; White Tabor, C.; Tabor, H.
S-adenosylmethionine synthetase (methionine adenosyltransferase) (Escherichia coli)
Methods Enzymol.
94
219-222
1983
Escherichia coli
brenda
Gilliland, G.L.; Markham, G.D.; Davies, D.R.
S-adenosylmethionine synthetase from Escherichia coli. Crystallization and preliminary X-ray diffraction studies
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1983
Escherichia coli
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S-Adenosylmethionine synthetase from Escherichia coli
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255
9082-9092
1980
Escherichia coli
brenda
Takusagawa, F.; Kamitori, S.; Markham, G.D.
Structure and function of S-adenosylmethionine synthetase: Crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi at 2.8.ANG. resolution
Biochemistry
35
2586-2596
1996
Escherichia coli (P0A817), Escherichia coli
brenda
Taylor, J.C.; Markham, G.D.
The bifunctional active site of S-adenosylmethionine synthetase. Roles of the active site aspartates
J. Biol. Chem.
274
32909-32914
1999
Escherichia coli (P0A817), Escherichia coli, Escherichia coli XL1-Blue (P0A817)
brenda
LeGros, H.L., Jr.; Halim, A.B.; Geller, A.M.; Kotb, M.
Cloning, expression, and functional characterization of the b regulatory subunit of human methionine adenosyltransferase (MAT II)
J. Biol. Chem.
275
2359-2366
2000
Escherichia coli, Homo sapiens, Homo sapiens (Q9NZL9)
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Corrales, F.J.; Perez-Mato, I.; Sanchez Del Pino, M.M.; Ruiz, F.; Castro, C.; Garcia-Trevijano, E.R.; Latasa, U.; Martinez-Chantar, M.L.; Martinez-Cruz, A.; Avila, M.A.; Mato, J.M.
Regulation of mammalian liver methionine adenosyltransferase
J. Nutr.
132
2377S-2381S
2002
Escherichia coli, Homo sapiens, Rattus norvegicus
brenda
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The active site loop of S-adenosylmethionine synthetase modulates catalytic efficiency
Biochemistry
41
9358-9369
2002
Escherichia coli (P0A817), Escherichia coli, Escherichia coli XL1-Blue (P0A817)
brenda
Taylor, J.C.; Markham, G.D.
Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling
Arch. Biochem. Biophys.
415
164-171
2003
Escherichia coli (P0A817)
brenda
Komoto, J.; Yamada, T.; Takata, Y.; Markham, G.D.; Takusagawa, F.
Crystal structure of the S-adenosylmethionine synthetase ternary complex: a novel catalytic mechanism of S-adenosylmethionine synthesis from ATP and Met
Biochemistry
43
1821-1831
2004
Escherichia coli (P0A817), Escherichia coli
brenda
Markham, G.D.; Reczkowski, R.S.
Structural studies of inhibition of S-adenosylmethionine synthetase by slow, tight-binding intermediate and product analogues
Biochemistry
43
3415-3425
2004
Escherichia coli (P0A817)
brenda
Driskell, L.O.; Tucker, A.M.; Winkler, H.H.; Wood, D.O.
Rickettsial metK-encoded methionine adenosyltransferase expression in an Escherichia coli metK deletion strain
J. Bacteriol.
187
5719-5722
2005
Escherichia coli, Rickettsia prowazekii, Rickettsia typhi
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Markham, G.D.; Takusagawa, F.; Dijulio, A.M.; Bock, C.W.
An investigation of the catalytic mechanism of S-adenosylmethionine synthetase by QM/MM calculations
Arch. Biochem. Biophys.
492
82-92
2009
Escherichia coli (P0A817)
brenda
Hu, H.; Qian, J.; Chu, J.; Wang, Y.; Zhuang, Y.; Zhang, S.
DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl-L-methionine production in Pichia pastoris
J. Biotechnol.
141
97-103
2009
Streptomyces spectabilis, Escherichia coli (P0A817), Escherichia coli, Saccharomyces cerevisiae (P19358)
brenda
Taylor, J.C.; Bock, C.W.; Takusagawa, F.; Markham, G.D.
Discovery of novel types of inhibitors of S-adenosylmethionine synthesis by virtual screening
J. Med. Chem.
52
5967-5973
2009
Escherichia coli (P0A817)
brenda
Yao, G.; Qin, X.; Chu, J.; Wu, X.; Qian, J.
Expression, purification, and characterization of a recombinant methionine adenosyltransferase pDS16 in Pichia pastoris
Appl. Biochem. Biotechnol.
172
1241-1253
2014
Escherichia coli
brenda
Sun, M.; Guo, H.; Lu, G.; Gu, J.; Wang, X.; Zhang, X.E.; Deng, J.
Lysine acetylation regulates the activity of Escherichia coli S-adenosylmethionine synthase
Acta Biochim. Biophys. Sin. (Shanghai)
48
723-731
2016
Escherichia coli (P0A817), Escherichia coli
brenda
Niu, W.; Cao, S.; Yang, M.; Xu, L.
Enzymatic synthesis of S-adenosylmethionine using immobilized methionine adenosyltransferase variants on the 50-mM scale
Catalysts
7
238
2017
Escherichia coli, Escherichia coli BL21 (DE3)
-
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Semi-rationally engineered variants of S-adenosylmethionine synthetase from Escherichia coli with reduced product inhibition and improved catalytic activity
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129
109355
2019
Escherichia coli (P0A817), Escherichia coli, Escherichia coli K12 (P0A817)
brenda