ライフサイエンスコーパス: dUMP

1 dUMP binding is similar in both proteins, except that th

2 ugs by inhibiting thymidylate synthase as 5F-dUMP.

3 mplex with the mechanism based inhibitor, 5F-dUMP, and cofactor.

4 bited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide.

5 e active conformation of a loop containing a dUMP-binding arginine.

6 ive nucleotides containing an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide

7 5-[N-(pazidobenzoyl)-3-aminoallyl]-dUMP (AB-dUMP).

8 dCMP gave results comparable to that with AB-dUMP at proximal nucleotide positions and provided new e

9 g was made that contained a butyl chain (ABU-dUMP) to assess the effect of the chain's hydrophobicity

10 e analog, 5-[N-(pazidobenzoyl)-3-aminoallyl]-dUMP (AB-dUMP).

11 alent bond between the catalytic Cys 195 and dUMP is present in both subunits.

12 s of TS H199A/N229D in complex with dCMP and dUMP confirmed that the position and orientation of boun

13 e, and the mutant enzyme binds both dCMP and dUMP tightly but does not methylate dCMP.

14 deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively.

15 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar Km values with the Asn 229 mutants,

16 ng site, the thymidylate analog [32P]5-azido-dUMP was specifically photocrosslinked to the active sit

17 Besides binding dUMP, this loop has a key role in stabilizing the closed

18 Since bound dUMP forms the binding surface against which the pterin

19 ng an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine

20 o activity for the dehalogenation of 5-bromo-dUMP, which requires correct orientation of dUMP against

21 ate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as

22 ants, V3L and V3F, have strongly compromised dUMP binding, with K(m,app) values increased by factors

23 ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, throug

24 nated aryl azide (FAB-dUMP) or diazirine (DB-dUMP) coupled to 5-aminoallyl deoxyuridine were incorpor

25 of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10

26 the nucleotide substrate, 2'-deoxyuridylate (dUMP), and that stabilize a beta-bulge in the protein.

27 e by cytidine deaminase, leading to elevated dUMP/dTMP ratios.

28 steady-state intermediate-containing enzyme, dUMP, and cofactor accumulated with Tyr 146 mutants, and

29 The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor

30 ne (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB-dUMP) coupled to 5-aminoallyl deo

31 site communication occurs not upon the first dUMP binding, but upon the second.

32 MP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformationa

33 nd to the mechanism-based inhibitor 5-fluoro-dUMP (FdUMP) and methylenetetrahydrofolate (CH2THF) have

34 ntrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows

35 and in determining the binding affinity for dUMP (in contrast, the N229(177)V mutation in Lactobacil

36 Ligand binding studies revealed that Kds for dUMP binding to two defective mutants, Ala216 and Leu216

37 coli is reduced by 200-fold while the Km for dUMP is increased 200-fold and the Km for folate increas

38 plain the 10(4)-fold decrease in kcat/Km for dUMP.

39 TSs except Thr216 TS exhibited kcat/Kms for dUMP that are 10(3)-10(4) times lower, relative to that

40 Steady-state values of K(m) for dUMP and k(cat) were not substantially different among t

41 ld type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate.

42 involved in all three known pathways to form dUMP.

43 y well and assist in proton abstraction from dUMP.

44 The deoxyribose in the thymidine comes from dUMP, which must first be dephosphorylated.

45 rotein catalyzed the conversion of dTMP from dUMP.

46 In M. thermophila, the dTMP was formed from dUMP and [methylene-2H2]-5,10-methylenetetrahydrosarcina

47 nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor an

48 ethylene intermediate is common to both HETM-dUMP and dTMP formation.

49 uced 5-(2-hydroxyethyl)thiomethyl-dUMP (HETM-dUMP).

50 With one analogous E. coli TS mutant, HETM-dUMP formation occurred upon occupancy of the first subu

51 te synthase, we found that the ratio of HETM-dUMP to dTMP varies as a function of CH(2)H(4)folate con

52 hree C-terminal mutants of L. casei TS, HETM-dUMP formation was consistent with a model in which prod

53 ine at this relative position is involved in dUMP binding; however, the data indicate that Ser216 has

54 his enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mut

55 Furthermore, an incorporated dUMP served as a productive 3'-primer terminus for subse

56 Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, w

57 UTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide

58 Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged

59 nd the dehalogenation of 5-bromo- and 5-iodo-dUMP.

60 u mass increase of TS after inhibition by IP-dUMP with no mass difference being detected for the TS m

61 consistent with covalent modification by IP-dUMP, which was confirmed by proteolytic digestion of th

62 ropynyl-2'-deoxyuridine 5'-monophosphate (IP-dUMP) is a mechanism-based, irreversible inactivator of

63 proposed for the covalent modification of IP-dUMP by Tyr94, which, unlike an earlier proposal, does n

64 ide (residues 89-96), each containing the IP-dUMP adduct, were observed.

65 MS/MS analysis of the IP-dUMP-endoAspN peptide identified a modified 3-residue da

66 F/S290G) in complex with active site ligands dUMP and CB3717.

67 , where these hydrogen bonds cannot be made, dUMP binds in a misoriented or more disordered fashion.

68 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor

69 late (methyleneTHF), a donor for methylating dUMP to dTMP in DNA synthesis, to 5-methyltetrahydrofola

70 imental evidence for a 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction w

71 ylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by N(5),N(10)-methyhlenetetrahydrofolate, forming

72 uct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate.

73 ylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate.

74 substrate 2'-deoxyuridine-5'-monophosphate (dUMP).

75 substrate 2'-deoxyuridine 5'-monophosphate (dUMP).

76 h its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined.

77 If their absence in TS N229D.dUMP persists in the ternary complex, it could explain t

78 oci in mammalian cells to counteract de novo dUMP incorporation into DNA.

79 is present, in addition to the nucleotides (dUMP, FdUMP, or dGMP), a Td of 72 degrees C is achieved

80 In the absence of dUMP, TS shows a major peak of unfolding at 45 degrees C

81 In the presence of increasing amounts of dUMP progressive changes in the size of each peak occur,

82 ssful synthesis of a variety of analogues of dUMP is described in which the substituents are introduc

83 ogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by ab

84 Thus, the specific binding of dUMP by TS results from occlusion of competing substrate

85 modynamic dissection of multisite binding of dUMP to E. coli TSase shows the nucleotide binds to the

86 ay crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus caseiTS mutant N229D

87 (hydroxymethyl)uracil (H) as consequences of dUMP misincorporation or thymine oxidation, respectively

88 C 2.1.1.45) (TS) catalyzes the conversion of dUMP to dTMP and is therefore indispensable for DNA repl

89 is by inhibiting the enzymatic conversion of dUMP to dTMP.

90 sn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ioniz

91 myces cerevisiae to dissect the influence of dUMP misincorporation into DNA as a contributing mechani

92 preferential methylation of dCMP instead of dUMP by this mutant.

93 There was no effect on the Km of dUMP, and only moderate effects on the Km of the cofacto

94 thase catalyzes the reductive methylation of dUMP to dTMP and is essential for the synthesis of DNA.

95 e synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemo

96 ylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeuti

97 ly mechanism is the deficient methylation of dUMP to dTMP and subsequent incorporation of uracil into

98 which catalyzes the reductive methylation of dUMP to dTMP using (R)-N(5),N(10)-methylene-5,6,7,8-tetr

99 which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication d

100 at for wild type TS-catalyzed methylation of dUMP, and some mutants (N229C and -A) catalyze methylati

101 :A base pairs arising by misincorporation of dUMP during DNA replication.

102 amination of cytosine or misincorporation of dUMP instead of dTMP [4] [5], and it is the primary acti

103 uracil (HmU) whereas the misincorporation of dUMP into DNA generates uracil (U), replacing the methyl

104 in ring of cofactor binds, misorientation of dUMP results in higher Km values for cofactor.

105 residue which normally H-bonds to the 4-O of dUMP but is not essential for activity.

106 red water appears to hydrogen bond to 4-O of dUMP.

107 -dUMP, which requires correct orientation of dUMP against Cys198.

108 hydrogen bonds constrain the orientation of dUMP in binary complexes with dUMP, and in ternary compl

109 s of R178 substitution on the orientation of dUMP; 10-15-fold increases in for R23I and R178T reflect

110 yme is thermally unfolded in the presence of dUMP, two separate temperature transitions are evident,

111 At a ratio of dUMP/TS of 100, a major peak predominates with an unfold

112 on from position 5 of the pyrimidine ring of dUMP.

113 The stabilization of dUMP, FdUMP, and dGMP binding to Escherichia coli thymid

114 ics and determined the crystal structures of dUMP complexes of three of the most active, uncharged si

115 ion of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from th

116 on of 3-MedUMP more efficiently than that of dUMP.

117 ects on catalysis, in addition to effects on dUMP binding.

118 attack of the active site cysteine of TS on dUMP.

119 Thymidylate synthase (TS) methylates only dUMP, not dCMP.

120 ase was inhibited in the presence of dUTP or dUMP.

121 he cofactor aids in ordering and positioning dUMP for catalysis.

122 of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP.

123 The free energy for productive dUMP binding, DeltaG(S), increases by at least 1 kcal/mo

124 encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate.

125 toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active

126 containing a single fluorescein-substituted dUMP analog as a lesion.

127 be methylated of a totally buried substrate dUMP.

128 chanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a conf

129 f the mutants bound the nucleotide substrate dUMP with only moderate loss of binding affinity, indica

130 nd C-5 of the pyrimidine moiety of substrate dUMP.

131 ic dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bo

132 Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild ty

133 tions (1000K(M)) of the competing substrate, dUMP.

134 f TS from P. carinii bound to its substrate, dUMP, and a cofactor mimic, CB3717, was determined to 2.

135 the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformationa

136 drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, p

137 Thus simply admitting dCMP into the dUMP binding site of TS is not sufficient for methylatio

138 Binding of the dUMP substrate abolishes this flexibility and stabilizes

139 allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene.

140 Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using

141 s well positioned to transfer hydride to the dUMP exocyclic methylene.

142 that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would b

143 enzyme produced 5-(2-hydroxyethyl)thiomethyl-dUMP (HETM-dUMP).

144 the enzyme form with both subunits bound to dUMP and CH(2)H(4)folate.

145 e binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterize

146 at the Asp side chain does not contribute to dUMP binding.

147 minase, catalyzing the conversion of dCMP to dUMP, is an important enzyme in the de novo synthesis of

148 to function as a dUTPase, converting dUTP to dUMP and inorganic pyrophosphate.

149 dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi.

150 dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DN

151 thyl group from methylenetetrahydrofolate to dUMP to form dTMP.

152 ism is ordered in the following manner, TS + dUMP --> TS x dUMP + (6R)-5,10-CH2-H4folate --> TS x dUM

153 of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resol

154 about 0.5 A further than in the wild-type TS-dUMP complex.

155 n specificity for methylation of dCMP versus dUMP.

156 is a mixed (noncompetitive) inhibitor versus dUMP.

157 the hydrogen-bonding network between water, dUMP and side-chains in the active-site cavity contribut

158 least 1 kcal/mol for each mutant, even when dUMP orientation and mobility in the crystal structure i

159 zes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very

160 oaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding si

161 ex with dUMP and CB3717, and in complex with dUMP alone are determined at 2.4 A, and 2.5 A resolution

162 tal structures of N229(177)A in complex with dUMP and CB3717, and in complex with dUMP alone are dete

163 In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (

164 rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been

165 is mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafol

166 ) and of the wild-type enzyme complexed with dUMP and THF.

167 xes with dUMP, and in ternary complexes with dUMP and the TS cofactor, 5,10-methylene-5,6,7,8-tetrahy

168 orientation of dUMP in binary complexes with dUMP, and in ternary complexes with dUMP and the TS cofa

169 network, without sterically interfering with dUMP binding.

170 the Escherichia coli TS mutant E60(58)Q with dUMP and the cofactor analog CB3717 and have determined

171 in the following manner, TS + dUMP --> TS x dUMP + (6R)-5,10-CH2-H4folate --> TS x dUMP x (6R)-5,10-

172 kinetically identifiable reaction step, TS x dUMP x (6R)-5,10-CH2H4folate --> (TS x dUMP x (6R)-5,10-

173 TS x dUMP + (6R)-5,10-CH2-H4folate --> TS x dUMP x (6R)-5,10-CH2H4folate --> TS x dTMP x H2folate --

174 TS x dUMP x (6R)-5,10-CH2H4folate --> (TS x dUMP x (6R)-5,10-CH2H4folate)*, likely represents reorie

175 Blockage of the alternative yjjG (dUMP phosphatase) pathway for deoxyribose-1-phosphate ge