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. 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 (EutT^WT/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutT^WT/Co relative to that of 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 for EutT^WT/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 of either His67, His75, Cys79, Cys80, or Cys83 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.

Figure 1

Abs spectra collected at 4.5 K (gray traces) and variable-temperature MCD spectra at 7 T of (A) Co(II)Cbl in the presence of EutT^WT/Co and ~1 mM MgATP and (B) free Co(II)Cbl. MCD features arising from 4c Co(II)Cbl are labeled with Greek letters. The feature highlighted by an asterisk in the MCD spectrum in (A) at ~14 000 cm⁻¹ is due to the presence of Co(II) bound to EutT.

Figure 2

Abs spectra collected at 4.5 K (black traces) and variable-temperature MCD spectra at 7 T of (A) EutT^WT reconstituted with Co(II) and (B) as-purified EutT^WT/Zn incubated with equimolar excess of CoCl₂. Gray arrows in (A) indicate the bands at which the VTVH-MCD data shown in Figure 3 were collected.

Figure 3

VTVH-MCD data collected at 724, 679.5, and 306.5 nm for EutT^WT/Co (colored dots). Data were obtained at temperatures of 2.2, 4.5, 8, 15, and 25 K as a function of magnetic fields ranging from 0 to 7 T. Theoretical fits are shown as solid black lines.

Figure 4

ΔMCD spectra of EutT^WT/Co and selected variants obtained by taking the difference between spectra collected at 4.5 and 25 K. The main features associated with the primary divalent metal binding site are highlighted by a dashed vertical line (a). The derivative feature arising from Co(II) bound non-specifically or to a different site is also indicated (b).

Figure 5

Abs (Top), CD (middle), and MCD (bottom) spectra of AdoCbl in the presence of EutT^WT/Co (blue) and free in solution (red). Spectra were collected at 4.5 K unless noted otherwise. The MCD spectrum of AdoCbl in the presence of EutT^WT/Co collected at 4.5 K (bottom, dashed blue line) was scaled by half to facilitate a comparison with the corresponding 25 K trace (bottom, solid blue line)

Figure 6

Low-energy region of ΔMCD spectra of (A) EutT^WT/Co incubated with an approximately equimolar amount of AdoCbl and (B) substrate-free EutT^WT/Co collected at 4.5, 8 and 15 K after subtracting the corresponding MCD spectra obtained at 25 K (MCD signal intensity decreases with increasing temperature). The positions of the main features from the primary divalent metal binding site are highlighted by dashed vertical lines.

Figure 7

High-energy region of ΔMCD spectra of (A) EutT^WT/Co incubated with AdoCbl in an approximate equimolar ratio and (B) substrate-free EutT^WT/Co collected at 4.5, 8 and 15 K after subtracting the corresponding MCD spectra obtained at 25 K (MCD signal intensity decreases with increasing temperature). The center position of the main derivative-shaped feature associated with Co(II) bound non-specifically or to a different site is highlighted by a dashed vertical line.

Figure 8

Proposed mechanism for the binding of the divalent metal ion and the Co(II)Cbl and ATP substrates to EutT, based on the results obtained in the present study. (i) In the absence of the divalent transition metal cofactor, EutT is unable to bind Co(II)Cbl due, possibly, to an improper architecture of the DMB binding pocket. (ii) Binding of the divalent transition metal cofactor by the C80 and C83 residues of both protein monomers causes a reorganization of the DMB binding pocket that is lined by residues of the HX₁₁CCX₂C(83) motif. (iii) The change in the architecture of the DMB binding pocket now allows Co(II)Cbl (and ATP) to bind to the enzyme active site.