EzCatDB: S00527
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DB codeS00527
RLCP classification3.133.90030.394 : Transfer
CATH domainDomain 13.40.50.300 : Rossmann foldCatalytic domain
E.C.2.7.4.9

CATH domainRelated DB codes (homologues)
3.40.50.300 : Rossmann foldS00547,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

Enzyme Name
UniProtKBKEGG

O05891P0A720
Protein nameThymidylate kinaseThymidylate kinasedTMP kinase
thymidine monophosphate kinase
thymidylate kinase
thymidylate monophosphate kinase
thymidylic acid kinase
thymidylic kinase
deoxythymidine 5'-monophosphate kinase
TMPK
thymidine 5'-monophosphate kinase
SynonymsEC 2.7.4.9
Thymidine monophosphate kinase
dTMP kinase
TMPK
EC 2.7.4.9
Thymidine monophosphate kinase
dTMP kinase
TMPK
RefSeqNP_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)
NP_415616.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_489366.1 (Protein)
NC_007779.1 (DNA/RNA sequence)

KEGG pathways
MAP codePathways
MAP00240Pyrimidine metabolism

UniProtKB:Accession NumberO05891P0A720
Entry nameKTHY_MYCTUKTHY_ECOLI
ActivityATP + dTMP = ADP + dTDP.ATP + dTMP = ADP + dTDP.
SubunitHomodimer.Homodimer.
Subcellular location

CofactorBinds 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.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProductsintermediates
KEGG-idC00305C00002C00364C00008C00363
CompoundMagnesiumATPdTMPADPdTDP
Typedivalent metal (Ca2+, Mg2+)amine group,nucleotideamide group,nucleotideamine group,nucleotideamide group,nucleotide
ChEBI18420
15422
17013
16761
18075

PubChem888
5957
9700
6022
164628

              
1n5iAUnboundUnboundBound:TMPUnboundUnboundUnbound
1n5jABound:_MGUnboundUnboundUnboundBound:TYDUnbound
1n5kAUnboundUnboundBound:TMPUnboundUnboundUnbound
1n5kBBound:_MGUnboundBound:TMPUnboundUnboundUnbound
1n5lAUnboundUnboundBound:TMPUnboundUnboundUnbound
1n5lBBound:_MGUnboundUnboundAnalogue:DPOBound:TYDUnbound
1g3uABound:_MGUnboundBound:TMPUnboundUnboundUnbound
1gsiABound:_MGUnboundBound:TMPUnboundUnboundUnbound
1gtvABound:_MGUnboundUnboundUnboundBound:TYDUnbound
1gtvBBound:_MGUnboundBound:TMPUnboundUnboundUnbound
1mrnABound:_MGAnalogue:T5A(ATP)Analogue:T5A(TMP)UnboundUnboundUnbound
1mrsABound:_MGUnboundAnalogue:5HUUnboundUnboundUnbound
1w2gAUnboundUnboundAnalogue:THMUnboundUnboundUnbound
1w2gBUnboundUnboundAnalogue:THMUnboundUnboundUnbound
1w2hAUnboundUnboundAnalogue:ATMUnboundUnboundUnbound
1w2hBUnboundUnboundAnalogue:ATM 1209Analogue:ATM 1210UnboundUnbound
4tmkAUnboundAnalogue:T5A(ATP)Analogue:T5A(TMP)UnboundUnboundUnbound
5tmpAUnboundAnalogue:Z5A(ATP)Analogue:Z5A(ATM)UnboundUnboundUnbound

Active-site residues
resource
literature [6], [7] & [15]
pdbCatalytic residuesCofactor-binding residuescomment
           
1n5iALYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1n5jALYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1n5kALYS 13;ARG  95;       
ASP 9;GLU 166(Mg2+ binding)
invisible 150-161
1n5kBLYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1n5lALYS 13;ARG  95;       
ASP 9;GLU 166(Mg2+ binding)
invisible 150-161
1n5lBLYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1g3uALYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1gsiALYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1gtvALYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1gtvBLYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1mrnALYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1mrsALYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
 
1w2gALYS 13;ARG  95;       
ASP 9;GLU 166(Mg2+ binding)
invisible 150-161
1w2gBLYS 13;ARG  95;ARG 153
ASP 9;GLU 166(Mg2+ binding)
invisible 156-160
1w2hALYS 13;ARG  95;       
ASP 9;       (Mg2+ binding)
invisible 150-167
1w2hBLYS 13;ARG  95;       
ASP 9;GLU 166(Mg2+ binding)
invisible 153-160
4tmkALYS 16;ARG 100;ARG 153
GLU 12(Mg2+ binding)
 
5tmpALYS 16;ARG 100;ARG 153
GLU 12(Mg2+ binding)
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[2]p.602
[6]p.3685
[7]p.14049-14050
[10]p.638-639, Fig.53
[12]p.97-98
[15]Fig.6

references
[1]
PubMed ID9253402
JournalNat Struct Biol
Year1997
Volume4
Pages595-7
AuthorsKenyon GL
TitleAZT monophosphate knocks thymidylate kinase for a loop.
[2]
PubMed ID9253404
JournalNat Struct Biol
Year1997
Volume4
Pages601-4
AuthorsLavie A, Vetter IR, Konrad M, Goody RS, Reinstein J, Schlichting I
TitleStructure of thymidylate kinase reveals the cause behind the limiting step in AZT activation.
Related PDB1tmk,2tmk
Related UniProtKBP00572
[3]
PubMed ID9256270
JournalNat Med
Year1997
Volume3
Pages836-7
AuthorsHazuda D, Kuo L
TitleFailure of AZT: a molecular perspective.
[4]
PubMed ID9256287
JournalNat Med
Year1997
Volume3
Pages922-4
AuthorsLavie A, Schlichting I, Vetter IR, Konrad M, Reinstein J, Goody RS
TitleThe bottleneck in AZT activation.
[5]
PubMed ID9461164
JournalNat Med
Year1998
Volume4
Pages132
AuthorsBalzarini J, Degreve B, De Clercq E
TitleImproving AZT efficacy.
[6]
PubMed ID9521686
JournalBiochemistry
Year1998
Volume37
Pages3677-86
AuthorsLavie A, Konrad M, Brundiers R, Goody RS, Schlichting I, Reinstein J
TitleCrystal 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 PDB3tmk
Related UniProtKBP00572
[7]
PubMed ID9826650
JournalProc Natl Acad Sci U S A
Year1998
Volume95
Pages14045-50
AuthorsLavie A, Ostermann N, Brundiers R, Goody RS, Reinstein J, Konrad M, Schlichting I
TitleStructural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase.
Related PDB4tmk,5tmp
Related UniProtKBP37345
[8]
PubMed ID10585390
JournalJ Biol Chem
Year1999
Volume274
Pages35289-92
AuthorsBrundiers R, Lavie A, Veit T, Reinstein J, Schlichting I, Ostermann N, Goody RS, Konrad M
TitleModifying human thymidylate kinase to potentiate azidothymidine activation.
Related PDB1e2q,1e2f,1e2e,1e2g
[9]
PubMed ID10666613
JournalActa Crystallogr D Biol Crystallogr
Year2000
Volume56
Pages226-8
AuthorsLi de la Sierra I, Munier-Lehmann H, Gilles AM, Barzu O, Delarue M
TitleCrystallization and preliminary X-ray analysis of the thymidylate kinase from Mycobacterium tuberculosis.
[10]
PubMed ID10873853
JournalStructure Fold Des
Year2000
Volume8
Pages629-42
AuthorsOstermann N, Schlichting I, Brundiers R, Konrad M, Reinstein J, Veit T, Goody RS, Lavie A
TitleInsights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.
[11]
PubMed ID11071809
JournalJ Mol Biol
Year2000
Volume304
Pages43-53
AuthorsOstermann N, Lavie A, Padiyar S, Brundiers R, Veit T, Reinstein J, Goody RS, Konrad M, Schlichting I
TitlePotentiating 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 PDB1e99,1e9a,1e9b
[12]
PubMed ID11469859
JournalJ Mol Biol
Year2001
Volume311
Pages87-100
AuthorsLi de la Sierra I, Munier-Lehmann H, Gilles AM, Barzu O, Delarue M
TitleX-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution.
Related PDB1g3u
[13]
PubMed ID11914484
JournalActa Crystallogr D Biol Crystallogr
Year2002
Volume58
Pages607-14
AuthorsUrsby T, Weik M, Fioravanti E, Delarue M, Goeldner M, Bourgeois D
TitleCryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals.
Related PDB1gsi,1gtv
[14]
PubMed ID12662932
JournalJ Mol Biol
Year2003
Volume327
Pages1077-92
AuthorsFioravanti E, Haouz A, Ursby T, Munier-Lehmann H, Delarue M, Bourgeois D
TitleMycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.
Related PDB1n5l,1n5k,1n5i,1n5j
[15]
PubMed ID12454011
JournalJ Biol Chem
Year2003
Volume278
Pages4963-71
AuthorsHaouz A, Vanheusden V, Munier-Lehmann H, Froeyen M, Herdewijn P, Van Calenbergh S, Delarue M
TitleEnzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism.
Related PDB1mrn,1mrs
[16]
PubMed ID15628853
JournalBiochemistry
Year2005
Volume44
Pages130-7
AuthorsFioravanti E, Adam V, Munier-Lehmann H, Bourgeois D
TitleThe crystal structure of Mycobacterium tuberculosis thymidylate kinase in complex with 3'-azidodeoxythymidine monophosphate suggests a mechanism for competitive inhibition.
Related PDB1w2g,1w2h

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.

createdupdated
2002-05-242009-02-26


Copyright: Nozomi Nagano, JST & CBRC-AIST
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Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
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Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
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