Probing the S-adenosylmethionine-binding site of rat guanidinoacetate methyltransferase. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases

Abstract

Most mammalian non-nucleic acid methyltransferases share three sequence motifs. To gain insight into the S-adenosyl-methionine (AdoMet)-binding site of guanidinoacetate methyltransferase, we mutated several conserved residues that are found in or near motifs I and II. Conversion of either of two glycine residues of motif I (Gly67 and Gly69) to an alanine resulted in an inactive enzyme. These enzymes, although having UV absorption, fluorescence and far-UV CD spectra virtually identical with those of the wild-type enzyme, seem to be conformationally different from the wild-type enzyme as judged by near-UV CD spectra and the extent of urea denaturation, and are apparently not capable of binding AdoMet. Mutation of Tyr136 of motif II to a valine resulted in a decrease in Kcat/Km values for substrates. Changing this residue to a phenylalanine caused only a minor change in Kcat/Km for AdoMet. This suggests that the aromatic side chain stabilizes the binding of AdoMet. Mutagenic changes of Glu89, which is the residue corresponding to the conserved acidic residue on the C-terminal side of motif I, indicated its contribution to AdoMet binding. These results are consistent with the idea that both motifs I and II are crucial in forming the AdoMet binding site of guanidinoacetate methyltransferase.

Escherichia coliTehB RequiresS-Adenosylmethionine as a Cofactor To Mediate Tellurite Resistance

“…We observed R h values ranging from 6.1 to 7.5 nm for TehB(Phe98Tyr), which suggests that this particular mutant either is more susceptible to aggregation or oligomerizes into a decamer. The other mutants showed no change with SAM and tellurite binding, which is in agreement with studies of motifs I and II of a rat non-nucleic acid methyltransferase examined by a variety of spectroscopic methods (22). The study observed only minor conformational changes with mutations to key residues and concluded that the SAM-binding site has strict residue requirements.…”

“…Aromatic rings are considered to be involved in binding SAM through cation-quadrapole interactions with the positively charged sulfonium moiety of SAM (13,31). The importance of tyrosine in motif II of rat guanidinoacetate methyltransferase (RGAMT) has already been demonstrated (22). In TehB, four aromatic residues were found in this region; they are Phe (Ϫ5 position with respect to Asp), Tyr (Ϫ1), Phe (ϩ1), and Phe (ϩ10).…”

Mapping and Molecular Modeling ofS-Adenosyl-l-methionine Binding Sites inN-Methyltransferase Domains of the Multifunctional Polypeptide Cyclosporin Synthetase

“…Considering the data together, it is evident that the cofactor interacts with homologous sequence elements within the cofactor-binding site of the N-MTase domains of distinct NRPS systems. Furthermore, both studies are in line with a study of rat guanidinoacetate MTase, which identified a Tyr residue within the AdoMet-binding motif II region of this enzyme as forming the covalent adduct with AdoMet (50,51). The recent resolution of the crystal structure of rat guanidinoacetate MTase revealed that this residue is proximal to the AdoMet binding pocket of this enzyme (52).…”

A Novel S-Adenosyl-l-methionine:Arsenic(III) Methyltransferase from Rat Liver Cytosol

“…These sequences conform to the general consensus for motif I (hh(D/E)hGXGXG), where h is a hydrophobic amino acid, X is any amino acid, and the position of glycine residues is strongly conserved (49). Motif I has been associated with the binding of AdoMet by the enzyme and mutation of the conserved glycine residues in motif I in guanidinoacetate methyltransferase abolished its catalytic activity (50). An acidic residue which contributes to the binding of AdoMet has been found between 17 and 19 residues C-terminal to the end of motif I (48,50).…”

“…Motif I has been associated with the binding of AdoMet by the enzyme and mutation of the conserved glycine residues in motif I in guanidinoacetate methyltransferase abolished its catalytic activity (50). An acidic residue which contributes to the binding of AdoMet has been found between 17 and 19 residues C-terminal to the end of motif I (48,50). In S-adenosyl-L-methionine:arsenic(III) methyltransferase and human Cyt19, aspartate was found 20 residues C-terminal to the end of motif I.…”