ライフサイエンスコーパス: AdoCbl

1 AdoCbl produced by R. sphaeroides was identified and qua

2 nts of cob(II)alamin generated with [5'-2H2]-AdoCbl in D2O in comparison with AdoCbl in H2O reveal tw

3 th [5'-2H2]-AdoCbl in H2O, and with [5'-2H2]-AdoCbl in D2O.

4 has now been measured in D2O, with [5'-2H2]-AdoCbl in H2O, and with [5'-2H2]-AdoCbl in D2O.

5 talyzes the exchange of tritium from [5'-3H]-AdoCbl with solvent.

6 dual functions; i.e., it tailors the active AdoCbl form of the cofactor and then transfers it direct

7 lonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangemen

8 e catalytic cycles of all adenosylcobalamin (AdoCbl)-dependent enzymes, as in each case catalysis is

9 dical propagation from an adenosylcobalamin (AdoCbl) to a pyridoxal 5'-phosphate (PLP) cofactor.

10 thine 4,5-aminomutase, an adenosylcobalamin (AdoCbl)- and pyridoxal L-phosphate (PLP)-dependent enzym

11 lmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangemen

12 yl-CoA mutase (MUT) is an adenosylcobalamin (AdoCbl)-requiring mitochondrial matrix enzyme that catal

13 4,5-aminomutase (OAM), an adenosylcobalamin (AdoCbl; coenzyme B(12))-dependent isomerase, employs a l

14 coenzymes for 5,6-LAM are adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP).

15 es and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor.

16 ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL).

17 lus leichmannii catalyzes adenosylcobalamin (AdoCbl)-dependent nucleotide reduction, as well as excha

18 ed to bring the cofactors adenosylcobalamin (AdoCbl) and pyridoxal-5'-phosphate (PLP) and the substra

19 metry and apparent Kd for adenosylcobalamin (AdoCbl) are dependent upon the relative concentrations o

20 spectrum is observed for adenosylcobalamin (AdoCbl) in water and ethylene glycol.

21 ydrase and the functional adenosylcobalamin (AdoCbl) analogue 5'-deoxy-3',4'-anhydroadenosylcobalamin

22 cteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucle

23 he lower ligand, which in adenosylcobalamin (AdoCbl) is 5,6-dimethylbenzimidazole, and in adenosylpse

24 air (RP) stabilization in adenosylcobalamin (AdoCbl)-dependent enzymes and (ii) the manifestation of

25 etalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogena

26 to biological activity of adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) is the Co-C bond cle

27 of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides.

28 the carbon-cobalt bond of adenosylcobalamin (AdoCbl) at a rate approximately 10(11)-fold faster than

29 zation of the kinetics of adenosylcobalamin (AdoCbl) binding by stopped-flow fluorescence spectroscop

30 te in the biosynthesis of adenosylcobalamin (AdoCbl) in many prokaryotes.

31 e de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C

32 e belongs to the class of adenosylcobalamin (AdoCbl)-dependent carbon skeleton isomerases and catalyz

33 d for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase.

34 ed in the biosynthesis of adenosylcobalamin (AdoCbl).

35 catalyze the formation of adenosylcobalamin (AdoCbl, coenzyme B(12)) from cobalamin and ATP.

36 ide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B(12)).

37 d for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B(12) ).

38 ulsed-laser photolysis of adenosylcobalamin (AdoCbl; coenzyme B(12)) in AdoCbl-dependent ethanolamine

39 hat delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (

40 of ethanolamine requires adenosylcobalamin (AdoCbl) as a cofactor, and, intriguingly, we also identi

41 lass II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor.

42 bl) is an analogue of the adenosylcobalamin (AdoCbl) coenzyme.

43 enzyme that utilizes the adenosylcobalamin (AdoCbl) cofactor to catalyze the rearrangement of methyl

44 reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has

45 nosyl radical pair in the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from

46 y, studied herein are the adenosylcobalamin (AdoCbl, also known as coenzyme B(12))-dependent diol deh

47 i, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucle

48 llus leichmannii utilizes adenosylcobalamin (AdoCbl) as a cofactor and, in addition to nucleotide red

49 (12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransfe

50 sm by which coenzyme B12 (adenosylcobalamin, AdoCbl)-dependent enzymes promote homolytic cleavage of

51 ,2-PD) in a coenzyme B12 (adenosylcobalamin, AdoCbl)-dependent fashion.

52 rate, and product in the adenosylcobalamin- (AdoCbl) dependent reaction of ethanolamine ammonia-lyase

53 es of Cbls is Ado-cobinamide (AdoCbi(+)), an AdoCbl derivative that lacks the tethered base 5,6-dimet

54 ding motif, the first to be discovered in an AdoCbl-dependent enzyme.

55 ase and acted as an in vitro inhibitor of an AdoCbl-dependent diol dehydratase.

56 l that the "unique" Abs spectra of MeCbl and AdoCbl, which differ considerably from the "typical" Abs

57 phosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed

58 resence of methylmalonyl-CoA mutase and ATP, AdoCbl is transferred from ATR to the acceptor protein i

59 r B12 biochemistry and renders coenzyme B12 (AdoCbl) so intriguingly suitable for enzymatic radical r

60 This protein, GlmES, binds AdoCbl stoichiometrically and neither the affinity for A

61 ared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and .CH(2)-Ado) generated

62 es rotation and a ~14 A translation to bring AdoCbl-initially positioned ~25 A away-into the active-s

63 studies reveal novel strategies employed by AdoCbl-dependent enzymes in the control of radical catal

64 sites in ATR are simultaneously occupied by AdoCbl.

65 r forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzy

66 r of the enzyme, 5'-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding.

67 e regulated by a single adenosyl cobalamine (AdoCbl)-responsive riboswitch.

68 reconstituted with both the native cofactor AdoCbl and its derivative methylcobalamin (MeCbl).

69 actor of 4 compared with the native cofactor AdoCbl.

70 y weakened affinity for the native cofactor, AdoCbl.

71 ylmalonic acidemia, resulting from defective AdoCbl binding.

72 te catalysis, the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor's Co-C bond is cleaved homolytically to

73 he mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 t

74 lamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl) have long fascinated chemists because of their c

75 coenzyme B(12) or 5'-deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for a number of isom

76 IcmF is a 5'-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skele

77 5'-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton re

78 of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl), in which the configuration of the N-glycosidic

79 pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and time-resolved prob

80 by significantly distorting the equilibrium AdoCbl structure.

81 ucture different from previously established AdoCbl riboswitches.

82 Compared with wild type, the affinity for AdoCbl is unchanged, but for the conversion of L-glutama

83 ichiometrically and neither the affinity for AdoCbl nor the turnover number depends upon protein conc

84 SR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding

85 quadrant of the molecule, as is the case for AdoCbl.

86 t to keep up with even the lowest demand for AdoCbl.

87 The mutations increased the K(m) for AdoCbl by 40- to 900-fold, while V(max) values varied fr

88 g that EutT employs a distinct mechanism for AdoCbl formation.

89 alue (245 microM compared to 0.54 microM for AdoCbl).

90 excited-state spectrum is also observed for AdoCbl bound to glutamate mutase.

91 8 +/- 0.01, which is 3-fold smaller than for AdoCbl in aqueous solution (0.23 +/- 0.01).

92 ibo)AdoCbl is 160-fold smaller than that for AdoCbl, and only 1/3 as much cob(II)alamin is produced a

93 A comparison of these results with those for AdoCbl in H2O revealed kH/kD of 1.6, 1.7, and 2.7, respe

94 e, only two of its active sites are used for AdoCbl synthesis and where binding of ATP to the vacant

95 that PduX is an L-threonine kinase used for AdoCbl synthesis.

96 , which attacks the 5'-carbon of ATP to form AdoCbl and inorganic triphosphate.

97 pKa of 3.7 for dimethylbenzimidazole in free AdoCbl.

98 the continuous-wave C-Co photolysis of free AdoCbl in 75% glycerol but have not done so in the therm

99 de that removal of the phosphoryl group from AdoCbl-5'-P is the last step in AdoCbl biosynthesis in s

100 of radical-based catalysis in this Class III AdoCbl-dependent enzyme.

101 In AdoCbl-depleted enzyme, formation of the external aldimi

102 denosylcobalamin (AdoCbl; coenzyme B(12)) in AdoCbl-dependent ethanolamine ammonia-lyase (EAL) from S

103 we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5'-

104 tion reaction from a primary CH(3)- group in AdoCbl-dependent methylmalonyl-CoA mutase shows the enzy

105 otated as cobY to reflect its involvement in AdoCbl biosynthesis.

106 l group from AdoCbl-5'-P is the last step in AdoCbl biosynthesis in serovar Typhimurium and that the

107 C-C bond angle are quite similar to those in AdoCbl.

108 ls the Rossmann domain, harboring the intact AdoCbl cofactor, is tilted toward the edge of the PLP bi

109 iguingly, we also identify an intercistronic AdoCbl riboswitch that has a predicted structure differe

110 tial for the conversion of AdopseudoCbl into AdoCbl, the cobamide needed for the catabolism of acetat

111 e incomplete corrinoid cobinamide (Cbi) into AdoCbl.

112 o their upper axial ligand, including MeCbl, AdoCbl, aquacobalamin (H(2)OCbl(+)), and vitamin B(12) (

113 In the proposed chemical mechanism, AdoCbl initiates the formation of substrate radicals, an

114 variants have sufficient activity to mediate AdoCbl synthesis in vivo.

115 steric effector dGTP (Km = 17 +/- 3 microM), AdoCbl (Km = 60 +/- 9 microM) and no external reductant.

116 me uses AdopseudoCbl as a substrate, but not AdoCbl.

117 phate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis.

118 ekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors

119 Salmonella obtains AdoCbl by assimilation of complex precursors, such as vi

120 In the absence of AdoCbl, EutX uses this structure to sequester EutV.

121 We demonstrate that association of AdoCbl to this riboswitch prevents formation of an intri

122 hydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected.

123 It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other pro

124 previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of AT

125 active site triggers ejection of 1 equiv of AdoCbl from an adjacent site.

126 ATR required for delivery of 1 equivalent of AdoCbl, from 4 to 1.

127 fectively compete with the back formation of AdoCbl.

128 in formed have been studied as a function of AdoCbl concentration and temperature.

129 at L-2-hydroxyglutarate-induced homolysis of AdoCbl occurs very rapidly, with a rate constant approac

130 , as well as exchange of the 5' hydrogens of AdoCbl with solvent.

131 visible stopped-flow kinetic measurements of AdoCbl homolysis obtained with deuterated substrates.

132 oupled to repositioning of the Ado moiety of AdoCbl from the eastern conformation to the northern con

133 (13)C introduced into the ribosyl moiety of AdoCbl.

134 proximately 7 A from its position as part of AdoCbl to a position where it is in contact with C1 of t

135 Photolysis of AdoCbl in EAL at 240 K leads to cob(II)alamin-5'-deoxyad

136 Photolysis of AdoCbl in EAL leads to a quantum yield at 10(-7) s for c

137 studied by using pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and

138 pha-ribazole-5'-P, alpha-RP), a precursor of AdoCbl.

139 arable with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicates that the hydrogen tra

140 ady-state conditions reveals the presence of AdoCbl but no cob(II)alamin.

141 In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes tr

142 uctures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its correspon

143 m the active site, precluding reformation of AdoCbl at the end of the turnover cycle.

144 roism (CD), and magnetic CD (MCD) spectra of AdoCbl are observed upon formation of holoenzyme, even i

145 ferase, which catalyzes the terminal step of AdoCbl synthesis.

146 73) and is required for de novo synthesis of AdoCbl (coenzyme B(12)).

147 dent enzymes or for the de novo synthesis of AdoCbl.

148 ith maximal activity that is 9.7% of that of AdoCbl itself, and a very high Km value (245 microM comp

149 in ring is considerably flatter than that of AdoCbl, with a fold angle of 11.7 degrees.

150 oRbl revealed a structure similar to that of AdoCbl.

151 ofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM.

152 (beta), which forms a part of the "base-off"-AdoCbl binding motif.

153 at synthesizes GkCblS ectopically makes only AdoCbl, even under growth conditions where the synthesis

154 e much smaller than those measured for other AdoCbl enzymes and model reactions for which hydrogen tu

155 erases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of

156 hosphorylate adenosylcobalamin-5'-phosphate (AdoCbl-5'-P), the product of the condensation of alpha-r

157 ulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO

158 thermolysis of their respective precursors, AdoCbl and 8-MeOAdoCbl.

159 ld, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the

160 Cloning and expression of the putative AdoCbl-dependent PCM with an alpha2beta2 heterotetrameri

161 rturbs the equilibrium between the reactant (AdoCbl-bound) state and the product (cob(II)alamin/5'-de

162 rch, the mechanism by which MMCM and related AdoCbl-dependent enzymes accelerate the rate for homolyt

163 of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation st

164 the L. innocua cblT and cblS genes restored AdoCbl synthesis from Cbi and alpha-R in a Salmonella en

165 op assembly pathway in cobC strains restored AdoCbl-5'-P synthesis from Cby in a cobC strain.

166 d carbon-cobalt bond cleavage of (alpha-ribo)AdoCbl is 160-fold smaller than that for AdoCbl, and onl

167 d in the Ado ligand is inverted [(alpha-ribo)AdoCbl], has been synthesized and its crystal structure

168 from samples prepared with [U-(13)C-ribosyl]-AdoCbl.

169 tant results from its inability to sequester AdoCbl for direct transfer to MCM.

170 ty; thus, variant proteins failed to support AdoCbl synthesis in vivo.

171 iant enzymes analyzed in this work supported AdoCbl biosynthesis in vivo.

172 that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of th

173 on, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with the

174 um and that the reaction is catalyzed by the AdoCbl-5'-P phosphatase (CobC) enzyme.

175 (alpha-RP) and adenosylcobinamide-GDP by the AdoCbl-5'-P synthase (CobS, EC 2.7.8.26) enzyme.

176 o PLP as an external aldimine and elicit the AdoCbl Co-C bond homolysis and the accumulations of cob(

177 of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the bet

178 ture, which is independent of changes in the AdoCbl structure, and specifically the Co-C bond length,

179 ic acid (DAB) induces rapid homolysis of the AdoCbl Co-C bond (781 s(-1), D-ornithine; 513 s(-1), DAB

180 ogether demonstrate unusual stability of the AdoCbl Co-C bond and that radical catalysis is coupled t

181 n, which is critical in the formation of the AdoCbl Co-C bond.

182 termediate required for the formation of the AdoCbl product.

183 otein that comprises the two subunits of the AdoCbl-dependent isobutyryl-CoA mutase flanking a G-prot

184 We report that the AdoCbl-binding riboswitch is part of a small, trans-acti

185 As compared to the AdoCbl data, however, Abs and MCD spectral changes for t

186 In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroa

187 min to cob(I)alamin that was adenosylated to AdoCbl by ATR.

188 to Co2+ to Co+, followed by adenosylation to AdoCbl.

189 ransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant.

190 th purified ATR can convert cob(II)alamin to AdoCbl in vitro.

191 inactive cobalamins, such as vitamin B12, to AdoCbl.

192 tly the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation.

193 homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.

194 site leads to the transfer of the high value AdoCbl product to the acceptor mutase.

195 s accelerated by 12 orders of magnitude when AdoCbl is bound to the protein active site, possibly thr

196 ingly, our previous studies showed that when AdoCbl is bound to the MMCM active site, no enzymatic pe

197 n as the global minimum, as is the case with AdoCbl itself.

198 th [5'-2H2]-AdoCbl in D2O in comparison with AdoCbl in H2O reveal twice as much cob(II)alamin in the

199 bstrate ATP to ATR that is fully loaded with AdoCbl leads to the ejection of 1 equivalent of the cofa

200 iol dehydrase at 0.02% of that observed with AdoCbl.

201 To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzi

202 by the sugar of F(2)CTP and the second with AdoCbl destruction.

203 f growth on similar medium supplemented with AdoCbl.