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

Abstract

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.

Spectroscopic Study of the EutT Adenosyltransferase from Listeria monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate

“…23, 24, 26 In the case of the Se EutT, the inability to convert Co(II)Cbi + to a 4C species was attributed to the presence of a putative binding pocket for the nucleotide loop of Co(II)Cbl to enhance its affinity for the native substrate and promote axial ligand dissociation. 22, 24 The EPR spectrum of Co(II)Cbl in the presence of the Lm EutT/MgATP complex reveals that in addition to the major 4C form, a minor 5C, base-off Co(II)Cbl species is generated (Figures 5 and S1). However, this species is unlikely to be catalytically relevant, as its relative population remained constant on addition of excess MgATP.…”

“…Thus, unlike Se EutT, Lm EutT does not appear to generate a 5C, base-off intermediate in the process of 4C Co(II)Cbl formation. 24 This difference in substrate activation mechanism is unsurprising given that Se EutT requires a divalent metal cofactor for 4C Co(II)Cbl formation, 22, 24 while Lm EutT lacks this cofactor.…”

“…18, 19 Unlike the PduO- and CobA-type ACATs, EutT from Salmonella enterica ( Se EutT) requires a divalent metal ion [Fe(II) or Zn(II)] for activity that is postulated to reside in a tetrahedral coordination environment provided by four Cys residues, which are part of a conserved HX 11 CCX 2 C(83) motif. 20–22 Although the three types of ACATs are non-homologous, they all generate a four-coordinate (4C) cob(II)alamin (Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate. This species is sufficiently potent to attack the 5’-carbon of ATP, so as to generate the Co−C(ATP) bond of AdoCbl.…”

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity

“…The lack of metal in Lm EutT is of interest, because in Se EutT Fe(II) somehow disrupts the coordination bond between DMB and the Co(II) ion of the ring. The resulting DMB-off form of the corrinoid substrate results in a Co(II) four-coordinate species whose redox potential is within physiological range and can be readily reduced to Co(I) cobalamin by dihydroflavins 22, 23, 45 . At present, it is unclear how Lm EutT generates the Co(II) four-coordinate species of the cobalamin in the absence of a metal ion.…”

“…To date, a structure of a EutT class ACAT has not been reported, but insights into the mechanism of the S. enterica enzyme have been published 8, 22, 23 . Unlike CobA and PduO, S. enterica EutT ( Se EutT) requires ferrous ions for optimal activity, but substantial activity is observed when Zn(II) ions substitute for Fe(II) ions.…”