Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:Co^Irrinoid Adenosyltransferase Precludes Adenosylation of Incomplete Co^IIrrinoids

Abstract Three distinct families of ATP:corrinoid adenosyltransferases (ACATs) exist that are capable of converting vitamin B₁₂ derivatives into coenzyme B₁₂ by catalyzing the thermodynamically challenging reduction of Co^IIrrinoids to form "supernucleophilic" Co^I intermediates. While the structures and mechanisms of two of the ACAT families have been studied extensively, little is known about the EutT enzymes beyond the fact that they exhibit a unique requirement for a divalent metal cofactor for enzymatic activity. In this study we have obtained compelling evidence that EutT converts cob(II)alamin into an effectively four-coordinate Co^II species so as to facilitate Co^II→Co^I reduction. Intriguingly, EutT fails to promote axial ligand dissociation from the substrate analogue cob(II)inamide, a natural precursor of cob(II)alamin. This unique substrate specificity of EutT has important physiological implications.