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Biochemistry v.56 no.2, 2017년, pp.364 - 375   SCI SCIE
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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

Pallares, Ivan G. (Department of Chemistry, University of WisconsinMadison, Madison, Wisconsin 53706, ) ; Moore, Theodore C. (Department of Microbiology, University of Georgia, Athens, Georgia 30602, ) ; Escalante-Semerena, Jorge C. (Department of Microbiology, University of Georgia, Athens, Georgia 30602, ) ; Brunold, Thomas C. (Department of Chemistry, University of WisconsinMadison, Madison, Wisconsin 53706, ) ;
  • 초록  

    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 HX 11 CCX 2 C(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 HX 11 CCX 2 C(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 HX 11 CCX 2 C(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. Graphic Abstract ACS Electronic Supporting Info


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