Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
twitter

MetaCyc Enzyme: flavin-dependent thymidylate synthase

Gene: thyX Accession Number: G-12113 (MetaCyc)

Synonyms: thymidylate synthase thyX

Species: Mycobacterium tuberculosis H37Rv

Subunit composition of flavin-dependent thymidylate synthase = [ThyX]4
         flavin-dependent thymidylate synthase subunit = ThyX

Summary:
The native apparent molecular mass was determined by gel filtration chromatography [Hunter08].

Thymidylate synthase is a key enzyme in cellular DNA synthesis. It converts dUMP to dTMP in the pathway for de novo biosynthesis of the DNA building block dTTP.

In 2002 it was discovered that some organisms do not posses the classical thymidylate synthase ( EC 2.1.1.45) encoded by gene thyA and also lacked gene tdk encoding the salvage enzyme thymidylate kinase. Instead, these organisms had an alternate, novel flavin-dependent thymidylate synthase (EC 2.1.1.148) encoded by gene thyX. Homologs of the thyX gene have been identified in some eubacterial, archael, slime mold and viral genomes (as shown in [Myllykallio02]). The genomes of some of these organisms contain only thyX, while others, such as Mycobacterium tuberculosis, contain both thyX and thyA, the reason for which is poorly understood but may be related to phase of the growth cycle. A ThyX motif RHRX7S has been identified in this protein family (Pfam Thy1, PF02511) (in [Park10, Myllykallio02, Lesley02, Graziani06, Leduc07] and reviewed in [Murzin02, Leduc04, Koehn10]).

ThyX showed no sequence homology with ThyA and it showed substantial differences from ThyA in crystal structure and reaction mechanism. All thymidylate synthases catalyze a reductive methylation involving the transfer of the methylene group of 5,10-methylenetetrahydropteroyl mono-L-glutamate to the C5-position of dUMP and a two electron reduction of the methylene group to a methyl group, producing the thymine moiety of dTMP. However, the reductive mechanism of the classical thymidylate synthase ThyA is distinctly different from ThyX. The ThyA reductive mechanism uses folate as both a 1-carbon donor and a source of reducing equivalents, producing dUMP and 7,8-dihydrofolate monoglutamate as products, and does not involve a flavin coenzyme or a third substrate. In contrast, the ThyX mechanism uses a flavin coenzyme as a source of reducing equivalents, which are derived from a reducing substrate. This NAD(P)H oxidase uses FAD to mediate hydride transfer in a methylation reaction that results in tetrahydropteroyl mono-L-glutamate as a product, rather than 7,8-dihydrofolate monoglutamate produced in the ThyA reaction. The chemical and kinetic mechanisms of ThyX and progress in the design of specific inhibitors are reviewed in [Koehn10, Koehn09].

The crystal structure of this enzyme has been determined at 2.0Å resolution [Sampathkumar05]. Interestingly, a subsequent crystallographic study intended to show a quaternary complex of ThyX-FAD-BrdUMP-NADP+ instead resulted in an unexpected binary ThyX-NADP+ complex. Implications for inhibitor design were discussed [Sampathkumar06].

The recombinant, six-His-tagged, wild-type enzyme from Mycobacterium tuberculosis H37Rv was cloned, expressed in Escherichia coli and purified. Recombinant, mutant enzymes were also prepared by site-directed mutagenesis and purified. Complementation studies of wild-type and mutant proteins expressed in an Escherichia coli ΔthyA mutant were also performed, along with biochemical assays for NADPH oxidation and tritium release assays for deprotonation of [5-3H]dUMP. These studies identified amino acid residues in an extended motif as essential to enzyme activity. Data also suggested that the mutant ThyX proteins formed tetramers similar to the wild-type protein. Implications for the reaction mechanism were discussed [Ulmer08].

Recombinant, His-tagged ThyX and ThyA enzymes from Mycobacterium tuberculosis H37Rv have also been expressed in Escherichia coli and characterized both kinetically and for their ligand binding preferences. Unlike some ThyA proteins that can bind their cognate mRNA coding sequences and inhibit their own translation, neither ThyA nor ThyX bound to their own mRNA, suggesting no regulation by autologous translational feedback [Hunter08].
The subunit apparent molecular mass was determined by SDS-PAGE [Ulmer08].

Map Position: [3,067,193 <- 3,067,945]

Molecular Weight of Polypeptide: 27.591 kD (from nucleotide sequence), 27.6 kD (experimental) [Ulmer08 ]

Molecular Weight of Multimer: 104.0 kD (experimental) [Hunter08]

Unification Links: Entrez-gene:887766 , Pride:P66930 , Protein Model Portal:P66930 , SMR:P66930 , String:83332.Rv2754c , UniProt:P66930

Relationship Links: InterPro:IN-FAMILY:IPR003669 , PDB:Structure:2AF6 , PDB:Structure:2GQ2 , PDB:Structure:3GWC , PDB:Structure:3HZG , Pfam:IN-FAMILY:PF02511 , Prosite:IN-FAMILY:PS51331

Gene-Reaction Schematic: ?

Credits:
Created 23-Jul-2010 by Fulcher CA , SRI International


Enzymatic reaction of: flavin-dependent thymidylate synthase

Synonyms: FAD-dependent thymidylate synthase

EC Number: 2.1.1.148

dUMP + a 5,10-methylene-tetrahydrofolate + NADPH + H+ <=> dTMP + a tetrahydrofolate + NADP+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: pyrimidine deoxyribonucleotides de novo biosynthesis III

Summary:
Note that the Enzyme Commission reaction equation for EC 2.1.1.148 does not include NAD(P)H as of this curation date. Here, NADPH is indicated as a cofactor for the Mycobacterium tuberculosis H37Rv enzyme [Hunter08, Ulmer08].

The dTMP synthesizing activities of ThyX and ThyA proteins were confirmed by identification of the reaction products by reversed phase HPLC. They were also verified by tritium release assay and in the case of ThyX, NADPH oxidation assay. Both enzymes showed standard Michaelis-Menten kinetics. For ThyX, the Km for NADPH was 47μM. It was noted that both ThyX and ThyA have relatively low catalytic rates for dTMP synthesis, with ThyX being the slowest [Hunter08].

For comparison, the Kms for ThyA were 4 μM for dUMP and 70μM for 5,10-methylenetetrahydropteroyl mono-L-glutamate. ThyA was inhibited by both F-dUMP, and the folate-based inhibitor 1843U89, which bound only weakly to ThyX [Hunter08].

Cofactors or Prosthetic Groups: FAD [Hunter08], NADPH [Hunter08]

Inhibitors (Unknown Mechanism): F-dUMP [Hunter08]

Kinetic Parameters:

Substrate
Km (μM)
Citations
dUMP
3.0
[Hunter08]
a 5,10-methylene-tetrahydrofolate
4.0
[Hunter08]


References

Graziani06: Graziani S, Bernauer J, Skouloubris S, Graille M, Zhou CZ, Marchand C, Decottignies P, van Tilbeurgh H, Myllykallio H, Liebl U (2006). "Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX." J Biol Chem 281(33);24048-57. PMID: 16707489

Hunter08: Hunter JH, Gujjar R, Pang CK, Rathod PK (2008). "Kinetics and ligand-binding preferences of Mycobacterium tuberculosis thymidylate synthases, ThyA and ThyX." PLoS One 3(5);e2237. PMID: 18493582

Koehn09: Koehn EM, Fleischmann T, Conrad JA, Palfey BA, Lesley SA, Mathews II, Kohen A (2009). "An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene." Nature 458(7240);919-23. PMID: 19370033

Koehn10: Koehn EM, Kohen A (2010). "Flavin-dependent thymidylate synthase: a novel pathway towards thymine." Arch Biochem Biophys 493(1);96-102. PMID: 19643076

Leduc04: Leduc D, Graziani S, Meslet-Cladiere L, Sodolescu A, Liebl U, Myllykallio H (2004). "Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea." Biochem Soc Trans 32(Pt 2);231-5. PMID: 15046578

Leduc07: Leduc D, Escartin F, Nijhout HF, Reed MC, Liebl U, Skouloubris S, Myllykallio H (2007). "Flavin-dependent thymidylate synthase ThyX activity: implications for the folate cycle in bacteria." J Bacteriol 189(23);8537-45. PMID: 17890305

Lesley02: Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, Stevens RC (2002). "Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline." Proc Natl Acad Sci U S A 99(18);11664-9. PMID: 12193646

Murzin02: Murzin AG (2002). "Biochemistry. DNA building block reinvented." Science 297(5578);61-2. PMID: 12029066

Myllykallio02: Myllykallio H, Lipowski G, Leduc D, Filee J, Forterre P, Liebl U (2002). "An alternative flavin-dependent mechanism for thymidylate synthesis." Science 297(5578);105-7. PMID: 12029065

Park10: Park M, Cho S, Lee H, Sibley CH, Rhie H (2010). "Alternative thymidylate synthase, ThyX, involved in Corynebacterium glutamicum ATCC 13032 survival during stationary growth phase." FEMS Microbiol Lett 307(2);128-34. PMID: 20636973

Sampathkumar05: Sampathkumar P, Turley S, Ulmer JE, Rhie HG, Sibley CH, Hol WG (2005). "Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0A resolution." J Mol Biol 352(5);1091-104. PMID: 16139296

Sampathkumar06: Sampathkumar P, Turley S, Sibley CH, Hol WG (2006). "NADP+ expels both the co-factor and a substrate analog from the Mycobacterium tuberculosis ThyX active site: opportunities for anti-bacterial drug design." J Mol Biol 360(1);1-6. PMID: 16730023

Ulmer08: Ulmer JE, Boum Y, Thouvenel CD, Myllykallio H, Sibley CH (2008). "Functional analysis of the Mycobacterium tuberculosis FAD-dependent thymidylate synthase, ThyX, reveals new amino acid residues contributing to an extended ThyX motif." J Bacteriol 190(6);2056-64. PMID: 18192395


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Thu Dec 18, 2014, biocyc14.