DB code: S00527

RLCP classification	3.133.90030.394 : Transfer
CATH domain	3.40.50.300 : Rossmann fold	Catalytic domain
E.C.	2.7.4.9
CSA
M-CSA
MACiE

CATH domain	Related DB codes (homologues)
3.40.50.300 : Rossmann fold	S00547 S00548 S00550 S00554 S00555 S00671 S00672 S00676 S00680 S00682 S00913 S00914 S00301 S00302 S00303 S00304 S00307 S00308 S00305 S00306 S00309 S00310 S00311 M00114 M00199 D00129 D00130 D00540 M00186

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	RefSeq
P0A720	Thymidylate kinase	TMPK EC 2.7.4.9 Thymidine monophosphate kinase dTMP kinase	NP_415616.1 (Protein) NC_000913.2 (DNA/RNA sequence) YP_489366.1 (Protein) NC_007779.1 (DNA/RNA sequence)
O05891	Thymidylate kinase	EC 2.7.4.9 Thymidine monophosphate kinase dTMP kinase TMPK	NP_217764.1 (Protein) NC_000962.3 (DNA/RNA sequence) NP_337873.1 (Protein) NC_002755.2 (DNA/RNA sequence) YP_006516723.1 (Protein) NC_018143.1 (DNA/RNA sequence)

KEGG enzyme name
dTMP kinase thymidine monophosphate kinase thymidylate kinase thymidylate monophosphate kinase thymidylic acid kinase thymidylic kinase deoxythymidine 5'-monophosphate kinase TMPK thymidine 5'-monophosphate kinase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
O05891	KTHY_MYCTU	ATP + dTMP = ADP + dTDP.	Homodimer.		Binds 1 magnesium ion per subunit. This ion is required for catalysis, binding to the active site transiently (at the TMP- binding site), and probably acting as a clamp between the phosphoryl donor and acceptor.
P0A720	KTHY_ECOLI	ATP + dTMP = ADP + dTDP.	Homodimer.

KEGG Pathways
Map code	Pathways	E.C.
MAP00240	Pyrimidine metabolism

Compound table
	Cofactors	Substrates		Products		Intermediates
KEGG-id	C00305	C00002	C00364	C00008	C00363
E.C.
Compound	Magnesium	ATP	dTMP	ADP	dTDP
Type	divalent metal (Ca2+, Mg2+)	amine group,nucleotide	amide group,nucleotide	amine group,nucleotide	amide group,nucleotide
ChEBI	18420 18420	15422 15422	17013 17013	16761 16761	18075 18075
PubChem	888 888	5957 5957	9700 9700	6022 6022	164628 164628

1n5iA	Unbound	Unbound	Bound:TMP	Unbound	Unbound	Unbound
1n5jA	Bound:_MG	Unbound	Unbound	Unbound	Bound:TYD	Unbound
1n5kA	Unbound	Unbound	Bound:TMP	Unbound	Unbound	Unbound
1n5kB	Bound:_MG	Unbound	Bound:TMP	Unbound	Unbound	Unbound
1n5lA	Unbound	Unbound	Bound:TMP	Unbound	Unbound	Unbound
1n5lB	Bound:_MG	Unbound	Unbound	Analogue:DPO	Bound:TYD	Unbound
1g3uA	Bound:_MG	Unbound	Bound:TMP	Unbound	Unbound	Unbound
1gsiA	Bound:_MG	Unbound	Bound:TMP	Unbound	Unbound	Unbound
1gtvA	Bound:_MG	Unbound	Unbound	Unbound	Bound:TYD	Unbound
1gtvB	Bound:_MG	Unbound	Bound:TMP	Unbound	Unbound	Unbound
1mrnA	Bound:_MG	Analogue:T5A(ATP)	Analogue:T5A(TMP)	Unbound	Unbound	Unbound
1mrsA	Bound:_MG	Unbound	Analogue:5HU	Unbound	Unbound	Unbound
1w2gA	Unbound	Unbound	Analogue:THM	Unbound	Unbound	Unbound
1w2gB	Unbound	Unbound	Analogue:THM	Unbound	Unbound	Unbound
1w2hA	Unbound	Unbound	Analogue:ATM	Unbound	Unbound	Unbound
1w2hB	Unbound	Unbound	Analogue:ATM_1209	Analogue:ATM_1210	Unbound	Unbound
4tmkA	Unbound	Analogue:T5A(ATP)	Analogue:T5A(TMP)	Unbound	Unbound	Unbound
5tmpA	Unbound	Analogue:Z5A(ATP)	Analogue:Z5A(ATM)	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [6], [7] & [15]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1n5iA	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1n5jA	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1n5kA	LYS 13;ARG 95;	ASP 9;GLU 166(Mg2+ binding)			invisible 150-161
1n5kB	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1n5lA	LYS 13;ARG 95;	ASP 9;GLU 166(Mg2+ binding)			invisible 150-161
1n5lB	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1g3uA	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1gsiA	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1gtvA	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1gtvB	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1mrnA	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1mrsA	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)
1w2gA	LYS 13;ARG 95;	ASP 9;GLU 166(Mg2+ binding)			invisible 150-161
1w2gB	LYS 13;ARG 95;ARG 153	ASP 9;GLU 166(Mg2+ binding)			invisible 156-160
1w2hA	LYS 13;ARG 95;	ASP 9; (Mg2+ binding)			invisible 150-167
1w2hB	LYS 13;ARG 95;	ASP 9;GLU 166(Mg2+ binding)			invisible 153-160
4tmkA	LYS 16;ARG 100;ARG 153	GLU 12(Mg2+ binding)
5tmpA	LYS 16;ARG 100;ARG 153	GLU 12(Mg2+ binding)

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[2]	p.602
[6]	p.3685
[7]	p.14049-14050
[10]	p.638-639, Fig.5	3
[12]	p.97-98
[15]	Fig.6

References
[1]
Resource
Comments
Medline ID
PubMed ID	9253402
Journal	Nat Struct Biol
Year	1997
Volume	4
Pages	595-7
Authors	Kenyon GL
Title	AZT monophosphate knocks thymidylate kinase for a loop.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID	9253404
Journal	Nat Struct Biol
Year	1997
Volume	4
Pages	601-4
Authors	Lavie A, Vetter IR, Konrad M, Goody RS, Reinstein J, Schlichting I
Title	Structure of thymidylate kinase reveals the cause behind the limiting step in AZT activation.
Related PDB	1tmk 2tmk
Related UniProtKB	P00572
[3]
Resource
Comments
Medline ID
PubMed ID	9256270
Journal	Nat Med
Year	1997
Volume	3
Pages	836-7
Authors	Hazuda D, Kuo L
Title	Failure of AZT: a molecular perspective.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID	9256287
Journal	Nat Med
Year	1997
Volume	3
Pages	922-4
Authors	Lavie A, Schlichting I, Vetter IR, Konrad M, Reinstein J, Goody RS
Title	The bottleneck in AZT activation.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	9461164
Journal	Nat Med
Year	1998
Volume	4
Pages	132
Authors	Balzarini J, Degreve B, De Clercq E
Title	Improving AZT efficacy.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID	9521686
Journal	Biochemistry
Year	1998
Volume	37
Pages	3677-86
Authors	Lavie A, Konrad M, Brundiers R, Goody RS, Schlichting I, Reinstein J
Title	Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) at 2.0 A resolution: implications for catalysis and AZT activation.
Related PDB	3tmk
Related UniProtKB	P00572
[7]
Resource
Comments
Medline ID
PubMed ID	9826650
Journal	Proc Natl Acad Sci U S A
Year	1998
Volume	95
Pages	14045-50
Authors	Lavie A, Ostermann N, Brundiers R, Goody RS, Reinstein J, Konrad M, Schlichting I
Title	Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase.
Related PDB	4tmk 5tmp
Related UniProtKB	P37345
[8]
Resource
Comments
Medline ID
PubMed ID	10585390
Journal	J Biol Chem
Year	1999
Volume	274
Pages	35289-92
Authors	Brundiers R, Lavie A, Veit T, Reinstein J, Schlichting I, Ostermann N, Goody RS, Konrad M
Title	Modifying human thymidylate kinase to potentiate azidothymidine activation.
Related PDB	1e2q 1e2f 1e2e 1e2g
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID	10666613
Journal	Acta Crystallogr D Biol Crystallogr
Year	2000
Volume	56
Pages	226-8
Authors	Li de la Sierra I, Munier-Lehmann H, Gilles AM, Barzu O, Delarue M
Title	Crystallization and preliminary X-ray analysis of the thymidylate kinase from Mycobacterium tuberculosis.
Related PDB
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID	10873853
Journal	Structure Fold Des
Year	2000
Volume	8
Pages	629-42
Authors	Ostermann N, Schlichting I, Brundiers R, Konrad M, Reinstein J, Veit T, Goody RS, Lavie A
Title	Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID	11071809
Journal	J Mol Biol
Year	2000
Volume	304
Pages	43-53
Authors	Ostermann N, Lavie A, Padiyar S, Brundiers R, Veit T, Reinstein J, Goody RS, Konrad M, Schlichting I
Title	Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP.
Related PDB	1e99 1e9a 1e9b
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID	11469859
Journal	J Mol Biol
Year	2001
Volume	311
Pages	87-100
Authors	Li de la Sierra I, Munier-Lehmann H, Gilles AM, Barzu O, Delarue M
Title	X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution.
Related PDB	1g3u
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID	11914484
Journal	Acta Crystallogr D Biol Crystallogr
Year	2002
Volume	58
Pages	607-14
Authors	Ursby T, Weik M, Fioravanti E, Delarue M, Goeldner M, Bourgeois D
Title	Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals.
Related PDB	1gsi 1gtv
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID	12662932
Journal	J Mol Biol
Year	2003
Volume	327
Pages	1077-92
Authors	Fioravanti E, Haouz A, Ursby T, Munier-Lehmann H, Delarue M, Bourgeois D
Title	Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.
Related PDB	1n5l 1n5k 1n5i 1n5j
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID	12454011
Journal	J Biol Chem
Year	2003
Volume	278
Pages	4963-71
Authors	Haouz A, Vanheusden V, Munier-Lehmann H, Froeyen M, Herdewijn P, Van Calenbergh S, Delarue M
Title	Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism.
Related PDB	1mrn 1mrs
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID	15628853
Journal	Biochemistry
Year	2005
Volume	44
Pages	130-7
Authors	Fioravanti E, Adam V, Munier-Lehmann H, Bourgeois D
Title	The crystal structure of Mycobacterium tuberculosis thymidylate kinase in complex with 3'-azidodeoxythymidine monophosphate suggests a mechanism for competitive inhibition.
Related PDB	1w2g 1w2h
Related UniProtKB

Comments
This enzyme, thymidylate kinase (TmpK), was very important in terms of the medicine for AIDS, azidothymidine (AZT) [1],[2],[3]. AZT is a prodrug that must be converted by cellular enzymes, such as thymidine kinase and TmpK, to the active triphosphate form [3]. According to the paper [12], the catalytic residues and magnesium binding site of the enzyme are so various and different between those from different organisms (bacteria, archaebacteria, eukaryote, and even mammal). The paper [7] mentioned that there are two types of TmpKs. Type I TmpKs stabilize the negative charge in the transition state by having the positively charged guanidinium group originating from the P-loop (yeast enzyme), whilst in the type II enzymes the guanidinium group originates from the LID region (E. coli enzyme). Usually, NMP kinases have catallytically essential residues in the LID region. Furthermore, TmpKs use only a few basic residues to interact with the transferred phosphoryl group, whereas other NMP kinases, such as adenylate kinase or uridylate kinase, use five [7]. For example, yeast TmpK appears to have the catalytic residues in the P-loop (Arg15, Lys19), whilst it lacks basic residues in its LID region [2], [6]. Moreover, the conserved arginine residue, Arg94 in yeast TmpK (Arg100 in E. coli enzyme) also appears to interact with both the gamma-phosphate of ATP and the phosphate of the nucleoside monophosphate (i.e., the transferred phosphate and the acceptor phosphate) [7]. In contrast, in the E. coli TmpK, Arg153 from the LID region and the conserved arginine, Arg100, as well as the lysine residue in the P-loop are involved in catalysis [7]. However, in the case of human TmpK, the arginine from the P-loop does not interact with the transferred phosphoryl group. Only the conserved arginine, Arg97, plays a role in catalysis, as well as the lysine from the P-loop, and acts as a clamp to bring the donor and acceptor [10]. The archaebacterial enzyme displayed very different features from the other TmpK enzymes [12], [14]. Whereas magnesium binding site for the human TmpK was located along the ATP-binding site, between the beta and gamma phosphate groups, the archaebacterial enzyme indicated the magnesium ion was positioned in the TMP-binding site [12], [14]. In addition, this ion plays an essential role in catalysis [14]. Firstly, the cation provides a strong electrostatic potential to attract the gamma-phosphate group of ATP sufficiently close to the alpha-phosphate group of TMP for phosphoryl transfer. Secondly, the crystal structures suggested that binding of ATP involves a direct coordination of the gamma-phosphate group of ATP onto the metal, which, locked tightly in the active site, is able to play the role of a clamp between the phosphoryl donor and acceptor [14]. As for the catalytic residues in the archaebacterial enzyme, Arg95 seems to neutralise the electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions, in concert with the magnesium ion [14]. Thus, catalysis by arginine can occur as long as the interaction with the transferred phosphate group is possible, regardless of where the arginine is located in the secondary structure [7]. The paper [7] also identified four different situations for catalytic arginines in phosphate-transferring enzymes; the catalytic arginine can be located (1) in the P-loop, (2) in other region (such as LID region), (3) in a different domain of a multidomain protein (see heterotrimeric G proteins), (4) in other protein capable of interacting with the phosphate-transferring protein (see Ras-RasGAP). This can explain why TmpK enzymes have only a few basic residues for the catalysis, whereas the other homologous NMP kinases have as many as five [7]. Furthermore, the paper [15] mentioned that the role of conserved serine residue (Ser99 in bacterial enzyme; PDB code, 1n5l, etc.) seems to be protonating the transferred PO3 group through Arg95 and Asp9. However, considering the structure with ligand, it seems unlikely.

Comments

This enzyme, thymidylate kinase (TmpK), was very important in terms of the medicine for AIDS, azidothymidine (AZT) [1],[2],[3]. AZT is a prodrug that must be converted by cellular enzymes, such as thymidine kinase and TmpK, to the active triphosphate form [3].
According to the paper [12], the catalytic residues and magnesium binding site of the enzyme are so various and different between those from different organisms (bacteria, archaebacteria, eukaryote, and even mammal). The paper [7] mentioned that there are two types of TmpKs. Type I TmpKs stabilize the negative charge in the transition state by having the positively charged guanidinium group originating from the P-loop (yeast enzyme), whilst in the type II enzymes the guanidinium group originates from the LID region (E. coli enzyme).
Usually, NMP kinases have catallytically essential residues in the LID region. Furthermore, TmpKs use only a few basic residues to interact with the transferred phosphoryl group, whereas other NMP kinases, such as adenylate kinase or uridylate kinase, use five [7].
For example, yeast TmpK appears to have the catalytic residues in the P-loop (Arg15, Lys19), whilst it lacks basic residues in its LID region [2], [6]. Moreover, the conserved arginine residue, Arg94 in yeast TmpK (Arg100 in E. coli enzyme) also appears to interact with both the gamma-phosphate of ATP and the phosphate of the nucleoside monophosphate (i.e., the transferred phosphate and the acceptor phosphate) [7]. In contrast, in the E. coli TmpK, Arg153 from the LID region and the conserved arginine, Arg100, as well as the lysine residue in the P-loop are involved in catalysis [7].
However, in the case of human TmpK, the arginine from the P-loop does not interact with the transferred phosphoryl group. Only the conserved arginine, Arg97, plays a role in catalysis, as well as the lysine from the P-loop, and acts as a clamp to bring the donor and acceptor [10].
The archaebacterial enzyme displayed very different features from the other TmpK enzymes [12], [14]. Whereas magnesium binding site for the human TmpK was located along the ATP-binding site, between the beta and gamma phosphate groups, the archaebacterial enzyme indicated the magnesium ion was positioned in the TMP-binding site [12], [14]. In addition, this ion plays an essential role in catalysis [14]. Firstly, the cation provides a strong electrostatic potential to attract the gamma-phosphate group of ATP sufficiently close to the alpha-phosphate group of TMP for phosphoryl transfer. Secondly, the crystal structures suggested that binding of ATP involves a direct coordination of the gamma-phosphate group of ATP onto the metal, which, locked tightly in the active site, is able to play the role of a clamp between the phosphoryl donor and acceptor [14]. As for the catalytic residues in the archaebacterial enzyme, Arg95 seems to neutralise the electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions, in concert with the magnesium ion [14].
Thus, catalysis by arginine can occur as long as the interaction with the transferred phosphate group is possible, regardless of where the arginine is located in the secondary structure [7]. The paper [7] also identified four different situations for catalytic arginines in phosphate-transferring enzymes; the catalytic arginine can be located
(1) in the P-loop,
(2) in other region (such as LID region),
(3) in a different domain of a multidomain protein (see heterotrimeric G proteins),
(4) in other protein capable of interacting with the phosphate-transferring protein (see Ras-RasGAP).
This can explain why TmpK enzymes have only a few basic residues for the catalysis, whereas the other homologous NMP kinases have as many as five [7].
Furthermore, the paper [15] mentioned that the role of conserved serine residue (Ser99 in bacterial enzyme; PDB code, 1n5l, etc.) seems to be protonating the transferred PO3 group through Arg95 and Asp9. However, considering the structure with ligand, it seems unlikely.

Created	Updated
2002-05-24	2009-02-26