Water-Mediated Carbon–Oxygen Hydrogen Bonding Facilitates S-Adenosylmethionine Recognition in the Reactivation Domain of Cobalamin-Dependent Methionine Synthase

Abstract

The C-terminal domain of cobalamin-dependent methionine synthase (MetH) has an essential role in catalyzing the reactivation of the enzyme following the oxidation of its cobalamin cofactor. This reactivation occurs through reductive methylation of the cobalamin using S-adenosylmethionine (AdoMet) as the methyl donor. Herein, we examine the molecular recognition of AdoMet by the MetH reactivation domain utilizing structural, biochemical, and computational approaches. Crystal structures of the Escherichia coli MetH reactivation domain in complex with AdoMet, the methyl transfer product S-adenosylhomocysteine (AdoHcy), and the AdoMet analogue inhibitor sinefungin illustrate that the ligands exhibit an analogous conformation within the solvent-exposed substrate binding cleft of the enzyme. AdoMet binding is stabilized by an intramolecular sulfur-oxygen chalcogen bond between the sulfonium and carboxylate groups of the substrate and by water-mediated carbon-oxygen hydrogen bonding between the sulfonium cation and the side chains of Glu1097 and Glu1128 that bracket the substrate binding cleft. AdoMet and sinefungin exhibited similar binding affinities for the MetH reactivation domain, whereas AdoHcy displayed an affinity for the enzyme that was an order of magnitude lower. Mutations of Glu1097 and Glu1128 diminished the AdoMet/AdoHcy binding selectivity ratio to approximately 2-fold, underscoring the role of these residues in enabling the enzyme to discriminate between the substrate and product. Together, these findings indicate that Glu1097 and Glu1128 in MetH promote high-affinity recognition of AdoMet and that sinefungin and potentially other AdoMet-based methyltransferase inhibitors can abrogate MetH reactivation, which would result in off-target effects associated with alterations in methionine homeostasis and one-carbon metabolism.

Crystallographic and Computational Characterization of Methyl Tetrel Bonding in S-Adenosylmethionine-Dependent Methyltransferases

“…These structures include the COMT/AdoMet/DNC/Mg 2+ , SMYD3/AdoMet/oxindole, and SMYD2/AdoMet/SGC BAY-598 complexes, as well as the G9A structure exhibiting a C∙∙∙O tetrel bond between AdoMet and a water molecule ( Figure 1 ). For the purposes of the QM calculations, AdoMet was represented as the sulfonium cation MeS + (Et) 2 , as previously reported [ 21 , 25 , 61 ]. The ability of the methyl group of this moiety to engage in a tetrel bond was first examined by computing its molecular electrostatic potential (MEP), as illustrated in Figure 2 .…”

Structural Basis for Stereoselective Dehydration and Hydrogen-Bonding Catalysis by the SAM-Dependent Pericyclase LepI

“…17 However, water molecules have rarely been observed as immediate hydrogen bond acceptor with SAM. 18 The water molecule W is also making a hydrogen bond with R295, which resides at the center of a ~500 Å 3 large cavity. This putative 6 active site cavity is outlined by both the catalytic domain (monomer A) and the dimerization domain from the other monomer (monomer B) within the homodimer unit (Figure 2d).…”

Hexacoordinated Tetrel‐Bonded Complexes between TF4 (T=Si, Ge, Sn, Pb) and NCH: Competition between σ‐ and π‐Holes

“…Closely related to these interactions are those in which the bridging atom comes from the tetrel family (C, Si, Ge, etc). [16][17][18][19][20][21] There is a rapidly growing literature [22][23][24][25][26][27] that has provided a wealth of insights into the chemical and physical phenomena that underlie tetrel bonds. This larger number of substituents obstructs a clear passage of an approaching nucleophile toward the tetrel atom, [8] which can inhibit the formation of such a bond or at the least require a good deal of deformation so as to clear a space for the Lewis base.…”

“…[9,10] Scores of tetrel bonds have been identified within protein structures, [11][12][13][14][15] and are implicated in the catalytic process of several enzymes. [16][17][18][19][20][21] There is a rapidly growing literature [22][23][24][25][26][27] that has provided a wealth of insights into the chemical and physical phenomena that underlie tetrel bonds. It is known for example, that the tetrel bond formed by a TR 4 molecule (T = tetrel atom) is strengthened by increasing electron-withdrawing capacity of the R substituent, [28][29][30] as well as increasing size of the T atom (i. e. C < Si < Ge).…”