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Supporting information
The Herbicide Ketoclomazone Inhibits 1-Deoxy-D-xylulose 5-Phosphate
Synthase in the 2-C-methyl-D-erythritol 4-Phosphate Pathway and Shows
Antibacterial Activity against Haemophilus Influenzae
Yukiko Matsue,a Hiroko Mizuno,a Takeo Tomita,a Tadao Asami,b,c Makoto
Nishiyama,a and Tomohisa Kuzuyamaa,*
a
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, and b RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Correspondence: [email protected]
Running head: Inhibition of DXP synthase by ketoclomazone
c
Present Address, Graduate School of Agricultural and Life Sciences, The University of
Tokyo 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657
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FIGURE LEGENDS
Figure S1. Expression and purification of the recombinant HiDXS enzyme.
Electrophoresis of the enzyme on an 8% SDS-polyacrylamide gel. Lanes: 1, low
molecular mass markers; 2, soluble protein after the induction; 3, fraction eluted with 60
mM imidazole from an Ni-NTA agarose column; 4, fraction eluted with 250 mM imidazole
from an Ni-NTA agarose column; 5, purified enzyme after gel filtration chromatography.
Figure S2. Kinetic analysis of ketoclomazone inhibition of E. coli DXP synthase.
A, Lineweaver-Burk plot for the inhibition of DXP synthase by ketoclomazone in the
presence of a fixed GAP concentration of 0.05 mM (≈ Kmpyruvate). The ketoclomazone
concentrations: 0 µM, circle; 50 µM, square; 100 µM, diamond; 200 µM, filled circle. B,
Secondary plot of the Lineweaver-Burk plot (A). The linear regression formula, the
correlation coefficient (R), and the formula for Ki are inserted. C, Lineweaver-Burk plot for
the inhibition of DXP synthase by ketoclomazone in the presence of a fixed pyruvate
concentration of 0.375 mM (≈ KmD-GAP). The ketoclomazone concentrations: 0 µM, circle;
100 µM, square; 200 µM, diamond; 500 µM, filled circle. D, Secondary plot of the
Lineweaver-Burk plot (C). Filled square, slope; filled circle, intercept. The linear
regression formulas, the correlation coefficients (R), and the formulas for Ki and Ki’ are
inserted.
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Figure S3. Multiple alignment of the amino acid sequences of DXP synthases.
This alignment was constructed using ClustalW ver. 1.82. Dots represent gaps
introduced for the optimization of the alignment. HiDXS, H. influenzae DXP synthase
(accession no. P45205); EcDXS, E. coli DXP synthase (accession no. P77488); AtDXS,
Arabidopsis thaliana DXP synthase (accession no. Q38854); DrDXS, Deinococcus
radiodurans (accession no. Q9RUB5).
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