Spectroscopic Studies of the EutT Adenosyltransferase from <i>Salmonella enterica</i>: Mechanism of Four-Coordinate Co(II)Cbl Formation

Pallares, Ivan G.; Moore, Theodore C.; Escalante‐Semerena, Jorge C.; Brunold, Thomas C.

doi:10.1021/jacs.5b11708

Cited by 12 publications

(70 citation statements)

References 44 publications

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“…3B). The spectral changes are similar to those reported previously for the Lactobacillus reuteri ATR (31) and are consistent with the formation of a 4-coordinate cob(II)alamin species lacking an axial water ligand (32,33). The EPR spectrum of cob(II)alamin (Fig.…”

Section: Gtp/gdp Are Allosteric Ligands Of Atr-purified C Metal-supporting

confidence: 89%

Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF

Zhu

Kitanishi

Twahir

et al. 2017

Journal of Biological Chemistry

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Edited by George N. DeMartinoIcmF is a 5-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor-and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. Herein, we demonstrate that the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the better characterized and homologous methylmalonyl-CoA mutase/G-protein chaperone system. Acyl-CoA mutases are a group of enzymes that utilize 5Ј-deoxyadenosylcobalamin (AdoCbl 4 or coenzyme B 12 ) as a cofactor for catalyzing carbon skeleton rearrangement reactions (1, 2). Isobutyryl-CoA mutase is a member of this enzyme family that is found in bacteria and catalyzes the reversible rearrangement of isobutyryl-and butyryl-CoA mutase ( Fig. 1A) (3). A chemically similar interconversion of methylmalonyl-CoA and succinyl-CoA is catalyzed by methylmalonyl-CoA mutase (MCM) (4), which is the only AdoCbl-dependent enzyme found in mammals. AdoCbl-dependent isomerization reactions are initiated by the homolytic cleavage of the cobalt-carbon bond of AdoCbl yielding cob(II)alamin and the working 5Ј-deoxyadenosyl radical, which initiates the chemical reaction by hydrogen atom abstraction from the substrate. AdoCbl thus serves as a latent radical reservoir (5), and substrate binding accelerates the homolytic cleavage rate in AdoCbl-dependent enzymes by a factor of ϳ10 12 (6, 7). At the end of each catalytic cycle, the 5Ј-deoxyadenosyl radical and cob(II)alamin recombine, thus regenerating the resting AdoCbl form of the cofactor (Fig. 1A). During catalytic turnover, the occasional loss of the 5Ј-deoxyadenosine moiety renders MCM prone to inactivation (8) and necessitates its dependence on a repair system for reentry into the catalytic cycle. Two proteins, adenosyltransferase (ATR) (9) and a G-protein chaperone (10), are needed for cofactor repair and reloading. The Methylobacterium extorquens orthologs of MCM, ATR and the G-protein (known as MeaB), have been characterized extensively (8,(11)(12)(13)(14)(15)(16)(17), and our understanding of the mechanism of cofactor repair derives primarily from studies on this system. The importance of the chaperonedependent cofactor loading and repair mechanisms is underscored by the existence of disease-causing mutations in both human ATR and the G-protein chaperone (18,19).IcmF is a naturally occurring fusion in which the G-protein chapero...

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Section: Gtp/gdp Are Allosteric Ligands Of Atr-purified C Metal-supporting

confidence: 89%

Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF

Zhu

Kitanishi

Twahir

et al. 2017

Journal of Biological Chemistry

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“…Previously, the mechanisms by which the CobA-type ACAT from Salmonella enterica , 31,32 the PduO-type human ACAT, 33 Lr PduO, 34 and the EutT-type ACAT from Salmonella enterica 35,36 can overcome the thermodynamically challenging Co(II) → Co(I)rrinoid reduction were investigated using magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. These studies led to the proposal that the ACAT/ATP complexes convert Co(II)rrinoids to effectively four-coordinate, square-planar species, thereby stabilizing the redox-active Co 3d z 2 -based molecular orbital (MO) and raising the reduction potential into the physiologically accessible range.…”

Section: Introductionmentioning

confidence: 99%

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri

et al. 2016

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The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi+). This process requires the formation of “supernucleophilic” Co(I)rrinoid intermediates in the enzyme active site that are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.

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“…Spectroscopic and kinetic characterizations of EutT variants with a single alanine substitution within this motif indicated that these residues are critical for the uptake of divalent metal ions [M(II)] and the formation of 4c Co(II)Cbl. 18,19,35 For example, EutT C80A was found to be unable to convert Co(II)Cbl to AdoCbl both in vivo and in vitro , which has been shown in a subsequent spectroscopic study to reflect the inability of this variant to bind Co(II)Cbl. 18 Similarly, EutT C83A and EutT H67A did not support ethanolamine catabolism, though low in vitro activity was observed.…”

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confidence: 89%

“…18,19,35 For example, EutT C80A was found to be unable to convert Co(II)Cbl to AdoCbl both in vivo and in vitro , which has been shown in a subsequent spectroscopic study to reflect the inability of this variant to bind Co(II)Cbl. 18 Similarly, EutT C83A and EutT H67A did not support ethanolamine catabolism, though low in vitro activity was observed. In contrast, the EutT C79A variant retained most of the activity displayed by the wild-type enzyme.…”

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confidence: 89%

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Evidence of a Tetrahedrally Coordinated Divalent Transition Metal Cofactor with Cysteine Ligation

et al. 2017

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The EutT enzyme from Salmonella enterica, a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes, requires a divalent transition metal ion for catalysis, with Fe(II) yielding the highest activity. EutT contains a unique cysteine-rich HX11CCX2C(83) motif (where H and the last C occupy the 67th and 83rd positions, respectively, in the amino acid sequence) not found in other ACATs and employs an unprecedented mechanism for the formation of adenosylcobalamin (AdoCbl). Recent kinetic and spectroscopic studies of this enzyme revealed that residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate (4c) cob(II)alamin [Co(II)Cbl] intermediate in the catalytic cycle. However, it remained unknown which, if any, of the residues in the HX11CCX2C(83) motif bind the divalent metal ion. To address this issue, we have characterized Co(II)-substituted wild-type EutT (EutTWT/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutTWT/Co relative to the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of 4c Co(II)Cbl. Our MCD data of EutTWT/Co also reveal that the Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Additional spectroscopic studies of EutT/Co variants possessing a single alanine substitution at either the His67, His75, Cys79, Cys80, or Cys83 position indicate that Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site.

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Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Mechanism of Four-Coordinate Co(II)Cbl Formation

Cited by 12 publications

References 44 publications

Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF

Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Evidence of a Tetrahedrally Coordinated Divalent Transition Metal Cofactor with Cysteine Ligation

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