Acid-base properties of .alpha.-ribazole and the thermodynamics of dimethylbenzimidazole association in alkylcobalamins

Brown, Kenneth L.; Hakimi, Janette M.; Nuss, Debra M.; Montejano, Yolanda D.; Jacobsen, Donald W.

doi:10.1021/ic00178a032

Cited by 88 publications

(74 citation statements)

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“…In contrast, the aquacobalamin form of I690C/G743C MetH(649-1227) is red at all temperatures, consistent with the very high affinity of Co 3ϩ in aquacobalamin for a coordinated base in the lower axial position (13). Surprisingly, the crosslinked mutant structure can assume a His-on conformation (see below).…”

Section: I690c/g743c Meth(649 -1227) Is Found In Both His-on and -Offmentioning

confidence: 60%

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

Datta¹,

Koutmos

Pattridge

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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B12-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-Lmethionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide crosslinking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme. multimodular protein ͉ protein conformation ͉ vitamin B12

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Section: I690c/g743c Meth(649 -1227) Is Found In Both His-on and -Offmentioning

confidence: 60%

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

Datta¹,

Koutmos

Pattridge

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…1D, MeCo(III)Cbl, Co(II)Cbl, and Co(I)Cbl differ in their preferred coordination states. In particular, the affinity of the cobalt for a nitrogen ligand in the lower axial position is directly proportional to the net positive charge on the cobalt (15)(16)(17). Entrance into the reactivation conformation is associated with dissociation of the histidine ligand from the Co(II)Cbl (18,19).…”

mentioning

confidence: 99%

Insights into the reactivation of cobalamin-dependent methionine synthase

Koutmos¹,

Datta²,

Pattridge³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every Ϸ2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from Sadenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH CT ) revealed a reactivation conformation that juxtaposes the cobalamin-and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH CT (S-SMetH CT ) that offer further insight into the reactivation of MetH. The structure of S-SMetH CT with cob(II)alamin and Sadenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of S-SMetH CT with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.corrinoid methyltransferase ͉ multimodular protein ͉ protein conformation ͉ cob(II)alamin coordination ͉ enzyme catalysis C obalamin-dependent methionine synthase (MetH) is a modular enzyme (1) that catalyzes methyl transfer from methyltetrahydrofolate (CH 3 -H 4 folate) to homocysteine (Hcy), forming tetrahydrofolate (H 4 folate) and methionine. The enzyme from Escherichia coli is one of the best-studied members of a large class of cobalamin-and corrinoid-dependent methyltransferases (2). In this class of enzymes, the cobalamin cofactor plays a crucial role in catalysis, acting both as a methyl donor and acceptor. During the MetH reaction cycle the methylcobalamin form of the cofactor [MeCo(III)Cbl] is demethylated by Hcy. The electrons of the carbon-cobalt bond remain on the cofactor producing cob(I)alamin [Co(I)Cbl]. Co(I)Cbl is subsequently remethylated by CH 3 -H 4 folate regenerating the MeCo(III)Cbl cofactor (Fig. 1A). However under aerobic conditions, the cobalamin cofactor is oxidized to an inactive cob(II)alamin [Co(II)Cbl] form about once in every 2,000 turnovers (3). To avoid accumulation of this inactive species, the enzyme undergoes a reductive reactivation, in which Co(II)Cbl is reduced to Co(I)Cbl with an electron supplied by flavodoxin (Fld), and Co(I)Cbl is then remethylated using S-adenosylmethionine (AdoMet) (4, 5). Reductive reactivation is a challenging reaction because the reduction potential of Co(II)Cbl in solution is out of the range of physiological reducing agents (6-8).MetH has 4 functional units th...

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“…[21,24] The acidbase properties of 2 À and 3 were studied by UV/Vis pH titration and pK a values of 5.5 and 6.2 were determined for 2-H ( Figure 3) and 3-H + , respectively. The pK a value of the former fragment is comparable to that of a-ribazole with a protonation at N-3 of the dimethylbenzimidazole base (pK a = 5.6), [27,28] and similarities between the major changes in the absorption spectra between 250 and 300 nm of these two compounds suggest that the protonation site is the same for 2 À . In summary, a one-pot synthesis of the AD and BC fragments of B 12 that combines photo-oxidative scission with an unprecedented subsequent demetallation, is described.…”

Section: Wwwchemeurjorgmentioning

confidence: 78%

One‐Pot Synthesis of the Metal‐Free AD and BC Fragments of Vitamin B₁₂

Zelder

Buchwalder

Oetterli

et al. 2010

Chemistry A European J

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Cobalamins are essential cofactors for the metabolism in mammals. [1, 2] Since fast proliferating cancer cells are strong consumers of vitamin B 12 (1; abbreviated as B 12 ) the development of B 12 -bioconjugates has recently attracted considerable attention. [3][4][5][6][7][8][9][10] On the other hand, the synthesis of vitamin B 12 analogues with metals other than cobalt (met-balamins) is still an unsolved chemical challenge. Several attempts failed to remove the cobalt ion directly from B 12 . [11,12] Although these potential antivitamins have been obtained from biotechnologically produced metal-free corrinoids followed by metal insertion, [13] thorough chemical and biological investigations of these metbalamin derivatives are still lacking.A metal-templated macrocyclisation of suitable precursors in the form of the AD and BC fragments of the natural product would offer an attractive chemical alternative. This strategy is closely related to the first total synthesis of B 12 achieved by the combined effort of Woodward et al. and Eschenmoser et al. [14][15][16][17][18] During the synthesis of the B 12 precursor cobyric acid, a "western" AD fragment was successfully joined with an "eastern" BC fragment. The building blocks had to be synthesised in a linear sequence of 37 steps for the former and 17 steps for the latter fragment, respectively. [16,17] In our endeavour aimed at the development of metbalamins for biological studies, we explored an efficient one-pot strategy for the cleavage and demetallation of B 12 into the related AD and BC fragments 2 À and 3 (Scheme 1). Kräutler et al. and others investigated extensively the photo-oxidation of corrinoids [19][20][21] and demonstrated the efficient removal of the cobalt ion from oxidatively cleaved hydrophobic cobyric acid heptamethylester derivatives (abbreviated as cobester). [21][22][23] In contrast to the cobester, the synthesis and demetallation of the related natural counterpart, an oxidised bicyclic B 12 derivative, has not yet been A C H T U N G T R E N N U N G achieved. Considering the strong solvent dependence on the reactivity of singlet oxygen towards corrinoids, [21] we were able to efficiently photo-oxidise dicyano-B 12 at the C-5 and C-15 meso-positions to the desired dicyano-tetraoxo-

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Acid-base properties of .alpha.-ribazole and the thermodynamics of dimethylbenzimidazole association in alkylcobalamins

Abstract: for performing the elemental analyses for antimony. They are thankful to H. W. Patrick for his skillful technical assistance.

Cited by 88 publications

References 5 publications

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

Insights into the reactivation of cobalamin-dependent methionine synthase

One‐Pot Synthesis of the Metal‐Free AD and BC Fragments of Vitamin B₁₂

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Acid-base properties of .alpha.-ribazole and the thermodynamics of dimethylbenzimidazole association in alkylcobalamins

Abstract: for performing the elemental analyses for antimony. They are thankful to H. W. Patrick for his skillful technical assistance.

Cited by 88 publications

References 5 publications

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

Insights into the reactivation of cobalamin-dependent methionine synthase

One‐Pot Synthesis of the Metal‐Free AD and BC Fragments of Vitamin B12

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Product

Resources

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One‐Pot Synthesis of the Metal‐Free AD and BC Fragments of Vitamin B₁₂