Dioldehydratase Binds Coenzyme B12 in the “Base-On” Mode: ESR Investigations on Cob(II)alamin

Abend, Andreas; Nitsche, Rainer; Bandarian, Vahe; Stupperich, Erhard; Rétey, János

doi:10.1002/(sici)1521-3773(19980316)37:5<625::aid-anie625>3.0.co;2-4

Cited by 53 publications

(28 citation statements)

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“…A possible functional advantage of the presence of a purinyl base in complete corrinoid cofactors in their H-bonding base-on constitution may therefore not be dismissed. Indeed, diol-dehydratase and cobamidedependent ribonucleotide reductase are two enzymes that depend upon adenosylcobamides and which have been discovered lately to carry their corrinoid cofactors in the base-on form (1,48,63).…”

Section: Discussionmentioning

confidence: 99%

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B₁₂and Factor A

et al. 2000

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The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co ␤ -cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 mol) of B 12 derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample of C. cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B 12 (Co ␤ -cyano-7؆-adeninylcobamide) (60%) and factor A (Co ␤ -cyano-7؆-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B 12 was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B 12 and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one-and two-dimensional 1 H, 13 C-, and 15 N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7؆-[N 6 -methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native "complete" B 12 cofactors the 7؆-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.

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Section: Discussionmentioning

confidence: 99%

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B₁₂and Factor A

et al. 2000

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“…EPR studies on other AdoCbl-requiring carbon skeleton mutases reveal that these class I enzymes also utilize a histidine as their axial ligand (1,6) as do most of the cobalamin-dependent methyltransferases thus far examined (37)(38)(39). In contrast, the AdoCbl enzymes catalyzing irreversible heteroatom rearrangements, typified by DD, use the DMB moiety of the cofactor as the axial ligand (7,8).…”

Section: Discussionmentioning

confidence: 99%

“…The x-ray structures of methylmalonyl-CoA (MMCoA) mutase (3,4) and EPR studies of cob(II)alamin (2) bound to other class I enzymes (1,5,6) revealed that in each case the dimethylbenzimidazole (DMB) moiety of the cofactor is replaced by a protein-derived histidine. On the other hand, in diol dehydrase (DD), typical of the class II enzymes, DMB remains ligated to 2 (7,8). Unique among the AdoCbl-requiring enzymes is ribonucleotide reductase, the only enzyme that does not catalyze a rearrangement reaction and in which C-Co homolysis occurs concomitant with the formation of a protein-based thiyl radical (9 -11).…”

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confidence: 99%

Binding of Cob(II)alamin to the Adenosylcobalamin-dependent Ribonucleotide Reductase fromLactobacillus leichmannii

Lawrence¹,

Gerfen²,

Sámano³

et al. 1999

Journal of Biological Chemistry

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The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5-triphosphates to 2-deoxynucleoside 5-triphosphates and uses coenzyme B 12 , adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2-deoxy-2-methylenecytidine 5-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.There are three classes of adenosylcobalamin (AdoCbl) 1 (Fig. 1, 1) requiring enzymes (1): those that catalyze reversible carbon skeleton rearrangements (class I), those that catalyze irreversible heteroatom elimination reactions (class II), and those that catalyze rearrangements involving migration of an amino group (class III). All three classes initiate their radicaldependent reactions by catalyzing carbon-cobalt bond homolysis. The mechanism(s) by which these enzymes catalyze C-Co homolysis of 1, the identification of the axial ligand trans to the C-Co bond that is cleaved, and its role in this process have recently been a topic of interest in many laboratories (2). The x-ray structures of methylmalonyl-CoA (MMCoA) mutase (3,4) and EPR studies of cob(II)alamin (2) bound to other class I enzymes (1, 5, 6) revealed that in each case the dimethylbenzimidazole (DMB) moiety of the cofactor is replaced by a protein-derived histidine. On the other hand, in diol dehydrase (DD), typical of the class II enzymes, DMB remains ligated to 2 (7,8). Unique among the AdoCbl-requiring enzymes is ribonucleotide reductase, the only enzyme that does not catalyze a rearrangement reaction and in which C-Co homolysis occurs concomitant with the formation of a protein-based thiyl radical (9 -11). The mechanism of homolysis and the identity of the axial ligand in this enzyme have not previously been addressed and are the subjects of this paper.Our laboratory has long been interested in the ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. In the presence of a nucleoside 5Ј-triphosphate (NTP) substrate, a reducing system, and a 2Ј-deoxynucleoside 5Ј-triphosphate (dNTP) allosteric effector, RTPR catalyzes homolysis of the C-Co bond of 1 to generate 2, 5Ј-deoxyadenosine, and a thiyl radical at Cys-408 of RTPR in a kinetically competent and concerted fashion (9,10,(12)(13)(14). This thiyl radical is then proposed to initiate the nucleotide reduction chemistry by abstraction of the 3Ј-hydrogen atom of the NTP (15). In this paper we report studies with a mechanism-based inhibitor of this protein, 2Ј-deoxy-2Ј-methylenecytidine 5Ј-triphosphate (MdCTP, 3). These studies in conjunction with [U- EXPERIMENTAL PROCEDURESMaterials and Methods-Alkaline phosphatas...

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“…This was the first crystallographic indication of the so-called 'base-on' mode of cobalamin binding to proteins, although this mode of cobalamin binding was indicated by EPR studies with 15 N-labeled coenzyme or its analogue [77,78]. In contrast, the X-ray structures of the methionine synthase cobalamin-binding domain [79], methylmalonyl-CoA mutase [74], and glutamate mutase [75], together with EPR studies on these enzymes [79][80][81], show that the DBI ligand is displaced from the cobalt atom and, instead, the imidazole group of a certain histidine residue in the cobalamin-binding motif part (DxHxxG) of proteins is ligated to the cobalt atom in these enzymes.…”

Section: Cobalamin Bindingmentioning

confidence: 99%

Radical catalysis of B 12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases

Toraya

2000

Cellular and Molecular Life Sciences (CMLS)

154

148

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Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals. Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions. The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (beta/alpha)8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent. Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis. The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure. The reactivating factors for diol and glycerol dehydratases have been identified. These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.

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Dioldehydratase Binds Coenzyme B12 in the “Base-On” Mode: ESR Investigations on Cob(II)alamin

Cited by 53 publications

References 9 publications

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B₁₂and Factor A

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B₁₂and Factor A

Binding of Cob(II)alamin to the Adenosylcobalamin-dependent Ribonucleotide Reductase fromLactobacillus leichmannii

Radical catalysis of B 12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases

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Dioldehydratase Binds Coenzyme B12 in the “Base-On” Mode: ESR Investigations on Cob(II)alamin

Cited by 53 publications

References 9 publications

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B12and Factor A

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B12and Factor A

Binding of Cob(II)alamin to the Adenosylcobalamin-dependent Ribonucleotide Reductase fromLactobacillus leichmannii

Radical catalysis of B 12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases

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Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B₁₂and Factor A

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B₁₂and Factor A