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 The stronger binding of the close analog 5F-dUMP to ThyX and its inhibitory properties compared with

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

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

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

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

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

8 5-[N-(pazidobenzoyl)-3-aminoallyl]-dUMP (AB-dUMP).

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

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

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

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

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

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

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

16 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar Km values with the Asn 229 mutants,

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

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

19 We find that human TS binds dUMP with ~9-fold entropically driven positive cooperati

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

43 carbinolamine intermediate used by ThyX for dUMP methylation.

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

45 involved in all three known pathways to form dUMP.

46 y well and assist in proton abstraction from dUMP.

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

48 rotein catalyzed the conversion of dTMP from dUMP.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

71 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor

72 stance of active site residues, to methylate dUMP.

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

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

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

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

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

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

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

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

81 eotide dTMP from deoxyuridine monophosphate (dUMP), making the enzyme necessary for DNA replication a

82 e utilization of deoxyuridine monophosphate (dUMP)/deoxy-5-fluorouridine monophosphate (5-FdUMP) as a

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

119 ctd that is considered as the main source of dUMP, the precursor of dTTP.

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

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

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

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

124 e used to characterize the thermodynamics of dUMP binding, with a focus on quantification of cooperat

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

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

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

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

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

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

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

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

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

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

135 be methylated of a totally buried substrate dUMP.

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

137 ed how ThyX recognizes the natural substrate dUMP in the N3-ionized form using an arginine, Arg199, i

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

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

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

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

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

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

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

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

146 us work on Escherichia coli TS revealed that dUMP substrate binds without cooperativity.

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

148 Binding of the dUMP substrate abolishes this flexibility and stabilizes

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

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

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

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

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

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

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

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

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

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

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

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

161 thyl group from methylenetetrahydrofolate to dUMP to form dTMP.

162 urther demonstrated that 2-OH-NQ, similar to dUMP, binds to ThyX in the ionized form, and the strong

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

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

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

166 n specificity for methylation of dCMP versus dUMP.

167 is a mixed (noncompetitive) inhibitor versus dUMP.

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

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

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

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

172 and its inhibitory properties compared with dUMP were explained by the stronger acidity of the uraci

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

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

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

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

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

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

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

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

181 network, without sterically interfering with dUMP binding.

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

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

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

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

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

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