Methyltransferases substrates

Fig. 10 SAM and clickable SAM analogs for labeling protein methyltransferase substrates...

Binda O, Boyce M, Rush JS et al (2011) A chemical method for labeling lysine methyltransferase substrates. Chembiochem 12 330-334... 

Peters W, Willnow S, Duisken M et al (2010) Enzymatic site-specific functionalization of protein methyltransferase substrates with alkynes for click labeling. Angew Chem Int Ed Engl 49 5170-5173... 

Uroporphyrinogen I (16c), a constitutional isomer of uroporphyrinogen III, also plays no direct role in porphyrin and corrin biosynthesis, but this unnatural substrate is methylated to give 17c10c f in the presence of SAM by the methyl transferase of some bacteria. A constitutional type I dihydroisobacteriochlorin can be obtained by methylation of uroporphyrinogen I with methylase Ml. Methyltransferase M1 is able to methylate the unnatural precorrin once more to give the trimethylpyrrocorphin type I.IOc 1... 

Catechol O-methyltransferase (COMT) is a widespread enzyme that catalyzes the transfer of the methyl group of S-adenosyl-l-methionine (AdoMet) to one of the phenolic group of the catechol substrate (Fig. 1). High COMT activity is found in the liver, kidney and gut wall... 

Catechol-O-Methyltransferase. Figure. 1 The basic function of COMT. Enzymatic O-methylation of the catechol substrate to 3-methoxy (major route) or 4-methoxy (minor route) products in the presence of Mg2+ and S-adenosyl-methionine (AdoMet). 

Catechol-O-Methyltransferase. Figure 2 Some substrates of COMT. 

With endogeneous pectic polysaccharides as substrates, the pectin methyhransferase activity was measured as radioactivity linked to oxalate-soluble polys x harides after extensive washing of microsomes with IM ethanolic NaCL Figure 2 shows that the rate of methylesterification of pectic substances was maximal on days 4 and 6 these maximum activities were observed within this period in at least five independent ejqjeriments. On the other hand, little activity was noted in young cells before day 2, and in old cells after day 9. In other words during the stationary phase the newly synthesised pectins remained unesterified because of the lack of pectin methyltransferase activity. 

In these experiments the pectin methyltransferase activity in vitro was generally only a few picokatal g" of protein, due mainly to the limited amount of SAM and endogeneous polysaccharide substrate. 

Figure 1 indicates that pectin methyltransferase (PMT) activity from freeze-thawed microsomes measured without exogenous substrate was maximal at neutral pH (6.5 to 7.5). When exogenous pectic substrates of various DE had been added, similar optimal neutral pH was observed, and the activity was slightly stimulated (1.2 to 1.8 times). A second optimal pH occured at pH 5.5, but in the presence of low methylated pectin (DE 0.1). As suggested by Lineweaver and Ballou [8] to explain the behaviour of another pectic enzyme -i.e. pectin methylesterase (PME), the mobility and the activity of PMT might be influenced by the presence of polyanionic substrates. On the other hand, the existence of several forms of pectin methyltransferase in flax microsomes might be responsible for such variations of the activity. 

Murray, Jr., E. D. and Clarke, S., Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase, J. Biol. Chem., 259, 10722, 1984. 

A common way to benefit from the ability to combine different molecular orbital methods in ONIOM is to combine a DFT or ab-initio description of the reactive region with a semi-empirical treatment of the immediate protein environment, including up to 1000 atoms. Due to the requirement for reliable semi-empirical parameters, as discussed in Section 2.2.1, this approach has primarily been used for non-metal or Zn-enzymes. Examples include human stromelysin-1 [83], carboxypeptidase [84], ribonucleotide reductase (substrate reaction) [85], farnesyl transferase [86] and cytosine deaminase [87], Combining two ab-initio methods of different accuracy is not common in biocatalysis applications, and one example from is an ONIOM (MP2 HF) study of catechol O-methyltransferase [88],... 

Deininger M, Szumlanski CL, Otter-ness DM et al. Purine substrates for human thiopurine methyltransferase. Bio-chem Pharmacol 1994 48 2135-2138. 

Woodson LC, Ames MM, Selassie CD et al. Thiopurine methyltransferase. Aromatic thiol substrates and inhibition by benzoic acid derivatives. Mol Pharmacol 1983 24 471-478. 

Biosynthesis is performed in one step by the enzyme L-histidine decarboxylase (HDC, E.C. 4.1.1.22). Histamine metabolism occurs mainly by two pathways. Oxidation is carried out by diamine oxidase (DAO, E.C. 1.4.3.6), leading to imidazole acetic acid (IAA), whereas methyla-tion is effected by histamine N-methyltransferase (HMT, E.C. 2.1.1.8), producing fe/e-methylhistamine (t-MH). IAA can exist as a riboside or ribotide conjugate. t-MH is further metabolized by monoamine oxidase (MAO)-B, producing fe/e-methylimidazole acetic acid (t-MIAA). Note that histamine is a substrate for DAO but not for MAO. Aldehyde intermediates, formed by the oxidation of both histamine and t-MH, are thought to be quickly oxidized to acids under normal circumstances. In the vertebrate CNS, histamine is almost exclusively methylated... 

The methyltransferases represent a relatively large number of enzymes that utilize the cofactor, S-adenosyl-L-methionine, in which the methyl group is bound to a positively charged sulfur, to transfer a methyl group to an oxygen, sulfur, or nitrogen atom in an appropriate substrate as shown in Figure 7.9 (8). 

FIGURE 7.12 Examples of thiols that are substrates for methyltransferases. 

Histone-lysine methyltransferases are chromatin-bound enzymes that catalyses the addition of methyl groups onto lysine or arginine residues of chromatin-bound H3 and H4 [151]. The methyl group is transferred enzymatically to the histone with S-adenosyl methionine as the methyl donor. Histone methylases have been isolated from HeLa S-3 cells [182], chick embryo nuclei [183], and rat brain chromatin [184]. The histone methyltransferases methylated H3 and H4 in nucleosomes [184]. Histone-lysine methyltransferase is a chromatin-bound enzyme [129,151]. Initial characterization of the Tetrahymena macronuclear H3 methyltransferase suggests that the enzyme has a molecular mass of 400 kDa. The enzyme preferred free histones rather than nucleosomes as substrate [138]. More recent studies have now... 

Mammalian G9a is a SET-domain histone methyltransferase that methylated H3 at Lys-9 and Lys-27 in vitro and at Lys-9 in vivo [196]. The consensus sequence for G9a appears to be TKXXARKS. G9a is dilferent from Suv39hl is several ways. G9a nuclear localization is distinct from that of Suv39hl, which locate to heterochromatic foci [197]. Suv39hlj2 double mutant mice lose H3 Lys-9 methylation at pericentromeric heterochromatic regions but broad methylation of chromatin remains. It is the latter that is lost in GPa-deficient cells [196]. G9a, molecular mass about 100 kDa, methylates free H3 and nucleosomal H3 with a preference for the former however, the presence of H1 stimulates the methylation of chromatin substrates. Suv39hl, molecular mass about 650 kDa, methylates free H3 and H3 in nucleosomes with equivalent efficiency, but when HI is present, methylation of chromatin substrates is lessened [198]. 

Human Set9 is a 50 kDa H3 methyltransferase that methylates Lys-4 of H3. The enzyme methylated free H3 but not H3 in chromatin substrates. There is evidence that Set9 may stimulate activated transcription [198]. Set9 has the SET domain but lacks the cysteine-rich (pre-SET and post-SET) domains. Disruption of Saccharomyces cerevisiae and Saccharomyces pombe Setl obliterates H3 methyl Lys-4 [199]. Thus this SET domain containing protein appears to be a H3 Lys-4 methyltransferase, catalyzing both di- and tri-methylation of H3 Lys-4 [155]. However, studies with recombinant Setl failed to show histone methyltransferase activity. It has been suggested that other associated proteins may be required for the Setl to be catalytically active [139,200]. Indeed, Setl is associated with several... 

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