Spectroscopic Studies of the <i>Salmonella enterica</i> Adenosyltransferase Enzyme <i>Se</i>CobA: Molecular-Level Insight into the Mechanism of Substrate Cob(II)alamin Activation

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

doi:10.1021/bi5011877

Cited by 13 publications

(38 citation statements)

References 64 publications

(167 reference statements)

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“…The 5c→4c Co(II)Cbl conversion yield achieved by EutT WT /Fe in the presence of excess MgATP largely exceeds the conversion yields reported for Se CobA and Lr PduO under similar conditions (~8% and 40% respectively). 38,39 …”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of co-substrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a “supernucleophilic” cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g. Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate “base-on” and “base-off” Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of “base-off” Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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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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“…These features (referred to as the δ-, λ-, and σ-bands, respectively) are characteristic of 4c Co(II)Cbl. 17 However, several additional weak MCD features are observed on the high-energy side of the δ-band that have no counterparts in the MCD spectra of other 4c Co(II)rrinoid species. These features are attributed to the high spin Co(II) center that is present in EutT WT /Co ( vide infra ).…”

Section: Resultsmentioning

confidence: 99%

“…In support of this proposal, it has been shown that amino acid substitutions in the active sites of the CobA- and PduO-type ACATs aimed at altering the relative positioning of the two substrates could cause a significant decrease in catalytic activity, despite the fact that the variants retained the ability to generate sizable amounts of 4c Co(II)Cbl. 16,17 …”

Section: Discussionmentioning

confidence: 99%

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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 Salmonella enterica Adenosyltransferase Enzyme SeCobA: Molecular-Level Insight into the Mechanism of Substrate Cob(II)alamin Activation

Cited by 13 publications

References 64 publications

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

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

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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