Recent studies have demonstrated that carbon-oxygen (CH···O) hydrogen bonds have important roles in S-adenosylmethionine (AdoMet) recognition and catalysis in methyltransferases. Here, we investigate noncovalent interactions that occur between the AdoMet sulfur cation and oxygen atoms in methyltransferase active sites. These interactions represent sulfur-oxygen (S···O) chalcogen bonds in which the oxygen atom donates a lone pair of electrons to the σ antibonding orbital of the AdoMet sulfur atom. Structural, biochemical, and computational analyses of an asparagine mutation in the lysine methyltransferase SET7/9 that abolishes AdoMet S···O chalcogen bonding reveal that this interaction enhances substrate binding affinity relative to the product S-adenosylhomocysteine. Corroborative quantum mechanical calculations demonstrate that sulfonium systems form strong S···O chalcogen bonds relative to their neutral thioether counterparts. An inspection of high-resolution crystal structures reveals the presence of AdoMet S···O chalcogen bonding in different classes of methyltransferases, illustrating that these interactions are not limited to SET domain methyltransferases. Together, these results demonstrate that S···O chalcogen bonds contribute to AdoMet recognition and can enable methyltransferases to distinguish between substrate and product.
Measuring the strength
of the hydrogen bonds between DNA base pairs
is of vital importance for understanding how our genetic code is physically
accessed and recognized in cells, particularly during replication
and transcription. Therefore, it is important to develop probes for
these key hydrogen bonds (H-bonds) that dictate events critical to
cellular function, such as the localized melting of DNA. The vibrations
of carbonyl bonds are well-known probes of their H-bonding environment,
and their signals can be observed with infrared (IR) spectroscopy.
Yet, pinpointing a single bond of interest in the complex IR spectrum
of DNA is challenging due to the large number of carbonyl signals
that overlap with each other. Here, we develop a method using isotope
editing and infrared (IR) spectroscopy to isolate IR signals from
the thymine (T) C2=O carbonyl. We use solvatochromatic studies
to show that the TC2=O signal’s position in the IR spectrum
is sensitive to the H-bonding capacity of the solvent. Our results
indicate that C2=O of a single T base within DNA duplexes experiences
weak H-bonding interactions. This finding is consistent with the existence
of a third, noncanonical CH···O H-bond between adenine
and thymine in both Watson–Crick and Hoogsteen base pairs in
DNA.
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.
The level of interest in probing the strength of noncovalent interactions in DNA duplexes is high, as these weak forces dictate the range of suprastructures the double helix adopts under different conditions, in turn directly impacting the biological functions and industrial applications of duplexes that require making and breaking them to access the genetic code. However, few experimental tools can measure these weak forces embedded within large biological suprastructures in the native solution environment. Here, we develop experimental methods for detecting the presence of a single noncovalent interaction [a hydrogen bond (Hbond)] within a large DNA duplex in solution and measure its formation enthalpy (ΔH f ). We report that introduction of a H-bond into the TC2�O group from the noncanonical nucleobase 2-aminopurine produces an expected decrease ∼10 ± 0.76 cm −1 (from ∼1720 cm −1 in Watson−Crick to ∼1710 cm −1 in 2-aminopurine), which correlates with an enthalpy of ∼0.93 ± 0.066 kcal/mol for this interaction.
The N-methyltransferase TylM1 from Streptomyces
fradiae catalyzes the final step in the biosynthesis of the
deoxyamino sugar mycaminose, a substituent of the antibiotic tylosin.
The high-resolution crystal structure of TylM1 bound to the methyl
donor S-adenosylmethionine (AdoMet) illustrates a
network of carbon–oxygen (CH···O) hydrogen bonds
between the substrate’s sulfonium cation and residues within
the active site. These interactions include hydrogen bonds between
the methyl and methylene groups of the AdoMet sulfonium cation and
the hydroxyl groups of Tyr14 and Ser120 in the enzyme. To examine
the functions of these interactions, we generated Tyr14 to phenylalanine
(Y14F) and Ser120 to alanine (S120A) mutations to selectively ablate
the CH···O hydrogen bonding to AdoMet. The TylM1 S120A
mutant exhibited a modest decrease in its catalytic efficiency relative
to that of the wild type (WT) enzyme, whereas the Y14F mutation resulted
in an approximately 30-fold decrease in catalytic efficiency. In contrast,
site-specific substitution of Tyr14 by the noncanonical amino acid p-aminophenylalanine partially restored activity comparable
to that of the WT enzyme. Correlatively, quantum mechanical calculations
of the activation barrier energies of WT TylM1 and the Tyr14 mutants
suggest that substitutions that abrogate hydrogen bonding with the
AdoMet methyl group impair methyl transfer. Together, these results
offer insights into roles of CH···O hydrogen bonding
in modulating the catalytic efficiency of TylM1.
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