ライフサイエンスコーパス: P450cam

1 P450cam from Pseudomonas putida is the best characterize

2 1) deep immersion in the membrane, and (2) a P450cam-like heme domain anchored to the membrane with o

3 Common conformational states among P450cam and homologous enzymes indicate that static and

4 nd compared for CYPs 1A2, 2B1, 2C9, 2E1, and P450cam.

5 mined that the ferryl forms of P450(BM3) and P450cam are protonated at physiological pH.

6 chloroperoxidase lie closer than do eNOS and P450cam on the truth diagram, it implies that the distal

7 ure just under the heme, in both P450cin and P450cam completely unfolds while this region is quite st

8 ces from cytochromes P450BM-3, P450terp, and P450cam, served as a test of the applicability of the su

9 ns between putidaredoxin reductase (Pdr) and P450cam and, thus, must form transient complexes with bo

10 inding and electron transfer between Pdx and P450cam, in a framework that allows for dynamical inform

11 region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydratio

12 show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformatio

13 containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed.

14 duced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO).

15 ed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state.

16 that Pdx would favor closed substrate-bound P450cam, which differs substantially from the open confo

17 rativity of potassium and camphor binding by P450cam, and also to influence the catalytic activity.

18 the surface of Pdx that can be recognized by P450cam.

19 nstrates that hydroxylation of substrates by P450cam in fact occurs by the formation and reaction of

20 oxidation of camphor and other substrates by P450cam.

21 binding results in conversion to the closed P450cam-C form.

22 stidine ligands in CN-SOR and the heme in CN-P450cam is directly compared by 14N ENDOR, while the axi

23 d in the closed conformation when ferrous-CO P450cam was titrated with reduced Pdx.

24 lied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner

25 ous dioxygen-bound P450 structures (CYP101A1/P450cam and CYP107A1/P450eryF) is proposed to participat

26 Cyt P450cam and cyt P450 1A2 showed 3-fold higher activity f

27 d on heme iron spin state, with low spin cyt P450cam giving a value 40-fold larger than high spin hum

28 We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on

29 Specifically, cytochrome (cyt) P450cam, cyt P40 1A2, and myoglobin in the array were ac

30 Cytochrome P450cam (CYP101) from Pseudomonas putida is unusual amon

31 Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that req

32 Cytochrome P450cam (CYP101Fe(3+)) regioselectively hydroxylates cam

33 Cytochrome P450cam (P450cam) is the terminal monooxygenase in a thr

34 Cytochrome P450cam was subjected to high pressures of 2.2 kbar, con

35 Although cytochrome P450cam from Pseudomonas putida, the archetype for all h

36 redoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida h

37 e (Pdr), putidaredoxin (Pdx), and cytochrome P450cam.

38 , camphor-free) and camphor-bound cytochrome P450cam (CYP101).

39 spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric st

40 e Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s.

41 cal role for potassium binding by cytochrome P450cam is to promote camphor binding even at the expens

42 ortant for dioxygen activation by cytochrome P450cam.

43 CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also

44 imately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P4

45 or the site-specific mutant D251N cytochrome P450cam (which affects proton transfer near the catalyti

46 The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein tw

47 eactions of substrate-free ferric cytochrome P450cam with peracids to generate Fe=O intermediates hav

48 s of camphor, dioxygen, and ferro-cytochrome P450cam to inject the "second" electron of the catalytic

49 annealing of the ternary ferrous cytochrome P450cam-O(2)-substrate complex.

50 en used to predict inhibitors for cytochrome P450cam and its L244A mutant.

51 ent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of

52 of the monooxygenase hemoprotein cytochrome P450cam.

53 Structural perturbations in cytochrome P450cam (CYP101) induced by the soluble fragment of cyto

54 he conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral pa

55 n many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in

56 n the biochemical cycle involving cytochrome P450cam.

57 e active site of microcrystalline cytochrome P450cam (CP450cam) in its resting state.

58 The camphor monoxygenase cytochrome P450cam (CYP101) requires potassium ion (K+) to drive fo

59 in the catalytic cycle of native cytochrome P450cam is the reduction of the dioxygen complex, which

60 ound state of the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putid

61 for corresponding derivatives of cytochrome P450cam and document significant and important differenc

62 applied to the cyanide adducts of cytochrome P450cam and its T252A and D251N site-directed mutants, b

63 Previous crystal structures of cytochrome P450cam complexed with its redox partner, putidaredoxin

64 al structures at 1.8A and 1.5A of cytochrome P450cam complexed with two synthetic molecular wires, D-

65 stantially different from that of cytochrome P450cam in that the B' helix, essential for substrate bi

66 The catalytic pathway of cytochrome P450cam is studied by means of a hybrid quantum mechanic

67 transfer into the active site of cytochrome P450cam is studied.

68 Fusion proteins of cytochrome P450cam with putidaredoxin (Pd) and putidaredoxin reduct

69 NO, and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the cor

70 sistent with analogous studies of cytochrome P450cam.

71 l based on the X-ray structure of cytochrome P450cam.

72 olution and compared with that of cytochrome P450cam.

73 or role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two differe

74 molecular dynamics simulations on cytochrome P450cam.

75 ve been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by gamma-irradiation at

76 hly investigated cytochrome P450, cytochrome P450cam.

77 e (camphor) binding to a protein (cytochrome P450cam).

78 topography more closely resembles cytochrome P450cam than cytochrome P450BM3.

79 ntaining component of the soluble cytochrome P450cam monooxygenase system from Pseudomonas putida, ha

80 Targeted dehydration of the cytochrome P450cam (CYP101) distal pocket through mutagenesis of a

81 x) with its redox partners in the cytochrome P450cam (CYP101) system was investigated by site-directe

82 The cytochrome P450cam active site is known to be perturbed by binding

83 electron transfer involved in the cytochrome P450cam catalytic cycle.

84 ent flavoprotein component of the cytochrome P450cam monooxygenase from Pseudomonas putida, has been

85 In the cytochrome P450cam-dependent monooxygenase system from Pseudomonas

86 involved in the binding of Pdx to cytochrome P450cam (CYP101).

87 A close orthologue to cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation

88 oxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating

89 m NADH-putidaredoxin reductase to cytochrome P450cam.

90 dioxygen bound form of wild type cytochrome P450cam were performed and the results analyzed to revea

91 Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into

92 of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other

93 In a racemic mixture of 2-ethylhexanol, P450cam produces 50% more (R)-2-ethylhexanoic acid than

94 Pdx was titrated into substrate-bound ferric P450cam, the enzyme shifted from the closed to the open

95 ted considerably higher Kd values for ferric P450cam and retained ca. 20% of the first electron trans

96 rison to the analogous derivatives of ferric P450cam.

97 alternative ET routes from Pdx(r) to ferric P450cam and a unique pathway to oxy-P450cam involving As

98 For P450cam, they are DeltaE(Q) = 1.84 mm/s and DeltaE(Q) =

99 dynamic regulation of enzymatic activity for P450cam in which the reduction potential is coupled to c

100 ibits redox-dependent binding affinities for P450cam and is thought to play an effector role in the m

101 Finally, we report the open form P450cam crystal structure with substrate bound, which su

102 nding residue, Gly248, in the substrate-free P450cam structure experiences a similar motion.

103 e increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold.

104 netic and modeling data we conclude that (i) P450cam-Pdx interaction is highly specific in part becau

105 ghly conserved carboxylate, aspartate-251 in P450cam in the distal helix I, participates in a series

106 e of the heme, about 15-20 A away, Asp251 in P450cam plays a critical role in a proton relay network

107 e species in the hydroxylation of camphor in P450cam.

108 R studies have failed to observe a change in P450cam conformation upon binding Pdx.

109 Observation of such large changes in P450cam suggests that substrate channel plasticity is a

110 to active-site residues may be different in P450cam and recently described mammalian P450 structures

111 ith ligands than the corresponding Gly248 in P450cam.

112 o the heme iron ligand, Cys357, is Leu358 in P450cam, whereas this residue is Ala in CYP101D1.

113 se sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and th

114 The corresponding residues in P450cam are Gly and Thr, respectively.

115 state in solution for the heme monooxygenase P450cam when bound to its natural redox partner, putidar

116 arbon monoxide into crystals of the nicotine-P450cam complex, to simulate molecular oxygen binding, p

117 This study examines the ability of P450cam to catalyze the formation of 2-ethylhexanoic aci

118 rspective to resolve how the conformation of P450cam depends on Pdx and ligand states.

119 show Pdx favors binding to the open form of P450cam.

120 hether Pdx favors the open or closed form of P450cam.

121 e ferric and ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different.

122 d their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP

123 Pdx and used to activate O(2) at the heme of P450cam.

124 e site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water

125 and, in the presence of saturating levels of P450cam, more effectively supported camphor hydroxylatio

126 High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates

127 Here we report X-ray structures of P450cam crystallized in the absence of substrate and at

128 Crystallographic studies of P450cam with (R)- or (S)-2-ethylhexanoic acid suggest th

129 loop unfolds in a manner similar to that of P450cam.

130 significant intermediate in the turnover of P450cam monooxygenase.

131 the substrate-free enzyme exists in the open P450cam-O conformation and that camphor binding results

132 ate population is observed for the D251N oxy-P450cam when the Pd complex is formed.

133 from Pd to the heme active site of D251N oxy-P450cam.

134 tly different from those for WT or D251N oxy-P450cam.

135 The D251N oxy-P450cam/Pd complex has a perturbed proton delivery mecha

136 the binding affinity of the mutants for oxy-P450cam was not substantially altered while the second E

137 presence of conformational substates in oxy-P450cam.

138 formational population redistribution of oxy-P450cam, along with the red-shifted electronic spectra,

139 the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putidaredoxin (Pd)

140 ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different.

141 Most significantly, in the oxy-P450cam complex Gly248 adopts a position midway between

142 o ferric P450cam and a unique pathway to oxy-P450cam involving Asp38; (iii) Pdx Trp106 is a key struc

143 Cytochrome P450cam (P450cam) is the terminal monooxygenase in a three-compon

144 (kcat = 30 min-1) was obtained with a PdR-Pd-P450cam construct in which the peptides TDGTASS and PLEL

145 E. coli cells expressing the PdR-Pd-P450cam fusion protein efficiently oxidize camphor to 5-

146 trength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predomin

147 rmation of a kinetically significant PdR/Pdx/P450cam complex.

148 dicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted b

149 Activity of the complete reconstituted P450cam system was measured, and kinetic parameters were

150 he crystal structure of oxidized and reduced P450cam complexed with its redox partner, putidaredoxin

151 plittings (P450(BM3), DeltaE(Q) = 2.16 mm/s; P450cam, DeltaE(Q) = 2.06 mm/s) are in good agreement wi

152 te that the effector role of Pdx is to shift P450cam toward the open conformation, which enables the

153 ctral studies indicate that the well-studied P450cam adopts the open conformation when its redox part

154 than P450s that melt at lower temperatures, P450cam and P450cin.

155 in eNOS resembles chloroperoxidase more than P450cam.

156 These data reveal that P450cam can dynamically visit an open conformation that

157 x and ferrous CO-bound P450cam, showing that P450cam remains closed.

158 dox partner, putidaredoxin (Pdx), shows that P450cam adopts the open conformation.

159 The P450cam monooxygenase from Pseudomonas putida consists o

160 As is the case for camphor, the P450cam exhibits stereoselectivity for binding (R)- and

161 e, which prevents electron transfer from the P450cam redox partner, Pdx.

162 sotope effect on product distribution in the P450cam reaction precludes a significant role for the P4

163 Extensive study of the P450cam active site has identified several residues that

164 ptors, by an increase in the activity of the P450cam domain upon addition of exogenous Pd, and by the

165 ence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant.

166 Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx).

167 to link the PdR to the Pd and the Pd to the P450cam domains.

168 A comparison between these P450cam and the new P450cin structures provides insights

169 Mutation of Ile-346, which corresponds to P450cam-Thr-252, an essential amino acid involved in dio

170 required to transfer electrons from NADH to P450cam, were constructed by fusing cDNAs encoding the t

171 ion proteins is electron transfer from Pd to P450cam.

172 which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the hem

173 uttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinet

174 that in solution, binding of reduced Pdx to P450cam does not favor the open conformation.

175 the proposal that binding of oxidized Pdx to P450cam opposes the open-to-closed transition induced by

176 e [2Fe-2S] containing putidaredoxin (Pdx) to P450cam.

177 tly to P420cam (Kd = 1.1 +/- 0.1 mM) than to P450cam (Kd = 4.6 +/- 0.2 mM), reflecting a weakened iro

178 to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to

179 nderstand differences between the two Pdx-to-P450cam ET events.

180 CKed into both the active sites of wild-type P450cam and its L244A mutant.

181 oxygen complex (Fe(II)-O2), of the wild-type P450cam and its mutants, D251N and T252A.

182 These results indicate that wild-type P450cam displays stereoselectivity toward 2-ethylhexanoi

183 us Pd, and by the high activity of wild-type P450cam when incubated with a PdR-Pd fusion protein.

184 e synthesized and found to inhibit wild-type P450cam.

185 silico and screened for binding to wild-type P450cam.

186 tude of 10 compared to 1.8 for the wild-type P450cam.

187 to be slightly larger than that of wild-type P450cam.

188 This in part explains why P450cam has such a strict requirement for Pdx.

189 A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering o

190 hat typical peroxidase chemistry occurs with P450cam and offer an explanation for the contrasting res

191 for generating highly oxidized species with P450cam should be valuable for their further characteriz