SUPPLEMENTAL DATA

Kinetic parameter values used in the model

(Km in µM, Vmax in µM/hr)

Parameter / Literature / Model / Reference[1] / Organism
DHFR
Km,DHF / 0.97 / 1.0 / (16) / Escherichia coli
Km,NADPH / 2.52 / 2.5 / (16) / Escherichia coli
Vmax / 500
ThyA
Km,dUMP / 1.1-1.2 / 1.0 / (4, 11, 13) / Escherichia coli
Km,5,10-CH2-THF
Vmax / 8.1-10 / 10
2250 / (11, 13) / Escherichia coli
ThyX
K cat / 0.35 / (8) / PBCV-1
Km,dUMP / 15 / 15 / (8) / PBCV-1
Km,5,10-CH2-THF
Vmax / 20 / 20
1200 / (8) / PBCV-1
KmNADPH / 40 / (8) / PBCV-1
DHFS – specific activity
Km dihydropteroate / 0.535
0.6 / Escherichia coli
FolM - specific activity (Vmax) / 0.083 / (7) / Escherichia coli
DHPR – specific activity
Vmax
Km DHF
Km NADPH / 5.2 / 225
0.5
4.0 / (20) / Thermus thermophilus
MTCH (positive direction is from 5,10-CH=THF to 10f-THF)
Km,5,10CH=THF / 80 / 80 / (14) / Methylobacterium extorquens
Spec activity
Vmax / 5,5 / 48000 / (2) / Escherichia coli
PGT
Km,10f-THF / 77.5 - 84.8 / 80 / (10) / Escherichia coli
Km,GARP / 8 - 12.2 / 10 / (10) / Escherichia coli
Spec activity
Vmax / 11.6 / 4000 / (10) / Escherichia coli
AICART
Km,10f-THF / 5.9
Km,AICARP / 50
Vmax / 10000
FTDF
Km10f-THF / 49 / 50 / (12) / Escherichia coli
Spec Activity
Vmax / 0.174 / 3000 / (12) / Escherichia coli
SHMT (positive direction is from THF to 5,10-CH2-THF)
Km,Ser / 150-300 / 300 / (5, 15) / Escherichia coli
Km,THF / 15-80 / 50 / (5, 15) / Escherichia coli
Spec Activity
Vmax / 13.6 / 6000 / (15) / Escherichia coli
MS
Km,5mTHF / 30-60 / 50 / (17, 18) / Escherichia coli
Km,Hcy / 16-69 / 50 / (18, 21) / Escherichia coli
Spec Activity / 3.8 / 5000 / (6) / Escherichia coli
MTD (5,10-CH2-THF  5,10-CH=THF)
Spec Activity
Vmax / 200 / 100000 / (2) / Escherichia coli
Km,5,10-CH2-THF / 26-45 / 30 / (1, 3) / Streptococcus faecium;Salmonella thyphimurium
Km,NADP+ / 21-92 / (1, 3) / Streptococcus faecium;Salmonella thyphimurium
MTHFR (5,10-CH2-THF  5mTHF)
Km,5,10-CH2-THF / 0.5-3.9 / 50 / (9, 19) / Escherichia coli
Km,NADH / 17-20 / 50 / (9, 19) / Escherichia coli
Vmax / 15

References

1.Albrecht, A. M., F. K. Pearce, and D. J. Hutchison. 1968. Methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A and its repressibility by serine. J Bacteriol 95:1779-89.

2.D'Ari, L., and J. C. Rabinowitz. 1991. Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli. J Biol Chem 266:23953-8.

3.Dalal, F. R., and J. S. Gots. 1967. Purification of 5,10-methylenetetrahydrofolate dehydrogenase from Salmonella typhimurium and its inhibition by purine nucleotides. J Biol Chem 242:3636-40.

4.Fantz, C., D. Shaw, W. Jennings, A. Forsthoefel, M. Kitchens, J. Phan, W. Minor, L. Lebioda, F. G. Berger, and H. T. Spencer. 2000. Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254. Mol Pharmacol 57:359-66.

5.Fu, T. F., E. S. Boja, M. K. Safo, and V. Schirch. 2003. Role of proline residues in the folding of serine hydroxymethyltransferase. J Biol Chem 278:31088-94.

6.Fujii, K., and F. M. Huennekens. 1974. Activation of methionine synthetase by a reduced triphosphopyridine nucleotide-dependent flavoprotein system. J Biol Chem 249:6745-53.

7.Giladi, M., N. Altman-Price, I. Levin, L. Levy, and M. Mevarech. 2003. FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli. J Bacteriol 185:7015-8.

8.Graziani, S., Y. Xia, J. R. Gurnon, J. L. Van Etten, D. Leduc, S. Skouloubris, H. Myllykallio, and U. Liebl. 2004. Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria Chlorella virus-1. J Biol Chem 279:54340-7.

9.Guenther, B. D., C. A. Sheppard, P. Tran, R. Rozen, R. G. Matthews, and M. L. Ludwig. 1999. The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat Struct Biol 6:359-65.

10.Inglese, J., D. L. Johnson, A. Shiau, J. M. Smith, and S. J. Benkovic. 1990. Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase. Biochemistry 29:1436-43.

11.Mahdavian, E., H. T. Spencer, and R. B. Dunlap. 1999. Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4. Arch Biochem Biophys 368:257-64.

12.Nagy, P. L., A. Marolewski, S. J. Benkovic, and H. Zalkin. 1995. Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli. J Bacteriol 177:1292-8.

13.Phan, J., E. Mahdavian, M. C. Nivens, W. Minor, S. Berger, H. T. Spencer, R. B. Dunlap, and L. Lebioda. 2000. Catalytic cysteine of thymidylate synthase is activated upon substrate binding. Biochemistry 39:6969-78.

14.Pomper, B. K., J. A. Vorholt, L. Chistoserdova, M. E. Lidstrom, and R. K. Thauer. 1999. A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1. Eur J Biochem 261:475-80.

15.Schirch, V., S. Hopkins, E. Villar, and S. Angelaccio. 1985. Serine hydroxymethyltransferase from Escherichia coli: purification and properties. J Bacteriol 163:1-7.

16.Shaw, D., J. D. Odom, and R. B. Dunlap. 1999. High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase. Biochim Biophys Acta 1429:401-10.

17.Taylor, R., and H. Weissbach. 1973. The Enzymes, 3rd Ed. (Boyer, P.D., ed.) 12:121-165.

18.Taylor, R., and H. Weissbach. 1971. Methods Enzymol 17B:379-388.

19.Trimmer, E. E., D. P. Ballou, M. L. Ludwig, and R. G. Matthews. 2001. Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: roles for aspartate 120 and glutamate 28. Biochemistry 40:6216-26.

20.Wilquet, V., M. Van de Casteele, D. Gigot, C. Legrain, and N. Glansdorff. 2004. Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus. J Bacteriol 186:351-5.

21.Zhou, Z. S., A. E. Smith, and R. G. Matthews. 2000. L-Selenohomocysteine: one-step synthesis from L-selenomethionine and kinetic analysis as substrate for methionine synthases. Bioorg Med Chem Lett 10:2471-5.

[1] If the reference is not indicated, the values where collected from BRENDA database.