Chemistry and enzymology of vitamin B12.

Radical catalysis in coenzyme B12-dependent isomerization (eliminating) reactions.

Radical carbon skeleton rearrangements: catalysis by coenzyme B12-dependent mutases.

This review describes enzymes that contain radicals and/or catalyze reactions with radical intermediates. Because radicals irreversibly react with dioxygen, most of these enzymes occur in anaerobic bacteria and archaea. Exceptions are the families of coenzyme B12- and S-adenosylmethionine (SAM)-dependent radical enzymes, of which some members also occur in aerobes. Especially oxygen-sensitive radical enzymes are the glycyl radical enzymes and 2-hydroxyacyl-CoA dehydratases. The latter are activated by an ATP-dependent one-electron transfer and act via a ketyl radical anion mechanism. Related enzymes are the ATP-dependent benzoyl-CoA reductase and the ATP-independent 4-hydroxybenzoyl-CoA reductase. Ketyl radical anions may also be generated by one-electron oxidation as shown by the flavin-adenine-dinucleotide (FAD)- and [4Fe-4S]-containing 4-hydroxybutyryl-CoA dehydratase. Finally, two radical enzymes are discussed, pyruvate:ferredoxin oxidoreductase and methane-forming methyl-CoM reductase, which ...

Radical enzymes in anaerobes.

The x-ray crystal structure of the pyridoxal-5′-phosphate (PLP), S-adenosyl-l-methionine (SAM), and [4Fe–4S]-dependent lysine-2,3-aminomutase (LAM) of Clostridium subterminale has been solved to 2.1-A resolution by single-wavelength anomalous dispersion methods on a l-selenomethionine-substituted complex of LAM with [4Fe–4S]2+, PLP, SAM, and l-α-lysine, a very close analog of the active Michaelis complex. The unit cell contains a dimer of hydrogen-bonded, domain-swapped dimers, the subunits of which adopt a fold that contains all three cofactors in a central channel defined by six β/α structural units. Zinc coordination links the domain-swapped dimers. In each subunit, the solvent face of the channel is occluded by an N-terminal helical domain, with the opposite end of the channel packed against the domain-swapped subunit. Hydrogen-bonded ionic contacts hold the external aldimine of PLP and l-α-lysine in position for abstraction of the 3-pro-R hydrogen of lysine by C5′ of SAM. The structure of the SAM/[4Fe–4S] complex confirms and extends conclusions from spectroscopic studies of LAM and shows selenium in Se-adenosyl-l-selenomethionine poised to ligate the unique iron in the [4Fe–4S] cluster upon electron transfer and radical formation. The chain fold in the central domain is in part analogous to other radical–SAM enzymes.

https://www.pnas.org/content/pnas/102/39/13819.full.pdf

The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterminale

Methylmalonyl-CoA mutase (MMCM) is an enzyme that utilizes the adenosylcobalamin (AdoCbl) cofactor to catalyze the rearrangement of methylmalonyl-CoA to succinyl-CoA. Despite many years of dedicated research, the mechanism by which MMCM and related AdoCbl-dependent enzymes accelerate the rate for homolytic cleavage of the cofactor's Co−C bond by ∼12 orders of magnitude while avoiding potentially harmful side reactions remains one of the greatest subjects of debate among B12 researchers. In this study, we have employed electronic absorption (Abs) and magnetic circular dichroism (MCD) spectroscopic techniques to probe cofactor/enzyme active site interactions in the Co3+Cbl “ground” state for MMCM reconstituted with both the native cofactor AdoCbl and its derivative methylcobalamin (MeCbl). In both cases, Abs and MCD spectra of the free and enzyme-bound cofactor are very similar, indicating that replacement of the intramolecular base 5,6-dimethylbenzimidazole (DMB) by a histidine residue from the enzyme acti...

Spectroscopic and computational studies on the adenosylcobalamin-dependent methylmalonyl-CoA mutase: evaluation of enzymatic contributions to Co-C bond activation in the Co3+ ground state.

The contribution of the active-site residue, Y89, to the trillion-fold acceleration of Co-carbon bond homolysis rate in the methylmalonyl-CoA mutase-catalyzed reaction has been evaluated by site-directed mutagenesis Conversion of Y89 to phenylalanine or alanine results in a 10(3)-fold diminution of k(cat) and suppression of the overall kinetic isotope effect The spectrum of the enzyme under steady-state conditions reveals the presence of AdoCbl but no cob(II)alamin Together, these results are consistent with homolysis becoming completely rate determining in the forward direction in the two mutants and points to the role of Y89 as a molecular wedge in accelerating Co-carbon bond cleavage

Tyrosine 89 accelerates Co-carbon bond homolysis in methylmalonyl-CoA mutase.

https://www.cell.com/trends/biochemical-sciences/pdf/S0968-0004(05)00116-7.pdf

Adenosyltransferase: an enzyme and an escort for coenzyme B12?

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.

Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Despite decades of research, the mechanism by which coenzyme B 12 (adenosylcobalamin, AdoCbl)-dependent enzymes promote homolytic cleavage of the cofactor's Co-C bond to initiate catalysis has continued to elude researchers. In this work, we utilized magnetic circular dichroism spectroscopy to explore how the electronic structure of the reduced B 12 cofactor (i.e., the post-homolysis product Co 2+ Cbl) is modulated by the enzyme methylmalonyl-CoA mutase. Our data reveal a fairly uniform stabilization of the Co 3d orbitals relative to the corrin π/π*-based molecular orbitals when Co 2+ Cbl is bound to the enzyme active site, particularly in the presence of substrate. Contrastingly, our previous studies (Brooks, A. J.; Vlasie, M.; Banerjee, R.; Brunold, T. C. J. Am. Chem. Soc. 2004, 126, 8167-8180.) showed that when AdoCbl is bound to the MMCM active site, no enzymatic perturbation of the Co 3+ Cbl electronic structure occurs, even in the presence of substrate (analogues). Collectively, these observations provide direct evidence that enzymatic Co-C bond activation involves stabilization of the post-homolysis product, Co 2+ -Cbl, rather than destabilization of the Co 3+ Cbl ground state.

Monica Vlasie

Papers

Spectroscopic and computational studies on the adenosylcobalamin-dependent methylmalonyl-CoA mutase: evaluation of enzymatic contributions to Co-C bond activation in the Co3+ ground state.

Tyrosine 89 accelerates Co-carbon bond homolysis in methylmalonyl-CoA mutase.

Adenosyltransferase: an enzyme and an escort for coenzyme B12?

Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Co-C bond activation in methylmalonyl-CoA mutase by stabilization of the post-homolysis product Co2+ cobalamin.