Methylation by SAM

Now the terminal amino group is methylated by SAM and the secondary amine cyclizes on to the pyridoxal imine to give an ami-nal. Decomposition of the aminal the other way round expels pyridoxamine and releases the salt of an electrophilic imine. 

Many health food stores now sell SAMe, a stabilized version of S-adenosylme-thionine. SAMe has been hypothesized to relieve depression because the synthesis of certain neurotransmitters requires methylation by SAM (see Chapter 47). This has led to the hypothesis that by increasing SAM levels within the nervous system, the biosynthesis of these neurotransmitters will be accelerated. This in turn might alleviate the feelings of depression. There have been reports in the literature indicating that this may occur, but its efficacy as an antidepressant must be confirmed. The major questions that must be addressed include the stability of SAMe in the digestive system and the level of uptake of SAMe by cells of the nervous system. 

Figure 1.7 Further examples of natural compounds IV-methylated by SAM lophocerine, galanthamine, lyngbyatoxi, and saframycin A.

Mammalian cells have three Ras proteins, Ha-Ras, Ki-Ras and N-Ras, which are present in most cell types. They are bound to the cytoplasmic surface of the plasma membrane by (i) a famesyl moiety bound by a thioether linkage to the C-terminal cysteine residue whose carboxyl group has been methylated by SAM (see) and (ii) a palmitoyl residue bound by a thioester linkage to a cysteine residue 2-5 residues from the C-terminus in the case of Ha- and N-Ras, or a polybasic domain containing six lysine residues in the case of Ki-Ras. 

As can be seen from the methylation step by SAM in Fig. 2, 2-methyl-6-phytylquinol is strongly preferred to its isomers. Thus, the main product is 2,3-dimethyl-5-phytylquinol which undergoes ringclosure (only verified in intact chloroplasts /5/) to form which is methylated by SAM to form rT. 

The reaction mechanism of PQ synthesis equals that of aT synthesis. The synthesis also occurs exclusively at the inner chloroplast envelope membrane /8/ (Fig. 4), however, it can be assumed that either prenylquinone is formed by its own enzyme garniture. 2-Methyl-6-nonaprenyl-(solanosyl-)qulnol is formed from homogentlsate plus nonaprenyl-(solanosyl-)PP. The quinol formed is then methylated by SAM to yield PQH /6/ (Fig. 4). Even if the sequence in PQ synthesis is clarified Homogentlsate — 2-Methyl-6—nonaprenylqulnol —PQH. no data are available for the synthesis of hydroxylated quinones... 

Figure 7.58 Catechols are the most common substrate for SAM. In this example the drug S-(-)-a-methyldopa is methylated by SAM to the 3-methoxy metabolite.The methyl transferase that catalyzes this process is called catechol 0-methyl transferase.

Phenazines — The phenazines are biosynthesized by the shikimic acid pathway, through the intermediate chorismic acid. The process was studied using different strains of Pseudomonas species, the major producers of phenazines. The best-known phenazine, pyocyanine, seems to be produced from the intermediate phenazine-1-carboxylic acid (PCA). Although intensive biochemical studies were done, not all the details and the intermediates of conversion of chorismic acid to PCA are known. In the first step, PCA is N-methylated by a SAM-dependent methyltransferase. The second step is a hydroxylative decarboxylation catalyzed by a flavoprotein monooxygenase dependent on NADH. PCA is also the precursor of phenazine-1-carboxamide and 1-hydroxyphenazine from Pseudomonas species. - - ... 

It is the role of jV5-methyl THF which is key to understanding the involvement of cobalamin in megaloblastic anaemia. The metabolic requirement for N-methyl THF is to maintain a supply of the amino acid methionine, the precursor of S-adenosyl methionine (SAM), which is required for a number of methylation reactions. The transfer of the methyl group from jV5-methyl THF to homocysteine is cobalamin-dependent, so in B12 deficiency states, the production of SAM is reduced. Furthermore, the reaction which brings about the formation of Ns-methyl THF from N5,N10-methylene THF is irreversible and controlled by feedback inhibition by SAM. Thus, if B12 is unavailable, SAM concentration falls and Ah -methyl THF accumulates and THF cannot be re-formed. The accumulation of AT-methyl THF is sometimes referred to as the methyl trap because a functional deficiency of folate is created. 

Important pathways requiring SAM include synthesis of epinephrine and of the 7-methylgua-nine cap on eukaryotic mRNA, Synthesis of SAM from methionine is shown in Figure T17-3. After donating the methyl group, SAM is converted to homocysteine and remethylated in a reaction catalyzed by N-methyl THF-homocysteine methyltransferase requirii both vitamin Bj2 and N-meth d-THF. The methionine produced is once again used to make SAM. 

Figure 15.4 Four compounds that are methylated either by SAM or methyl FH4 (CH3 FH4). The processes are (i) cytidine bases in DNA (11) methylatlon of deoxyUMP to produce deoxythymidine monophosphate (111) formation of phosphatidylcholine from phos-phatldylethanolamlne (Chapter 17) (Iv) methylatlon of a protein In myelin. The base cytidine Is methylated In DNA to produce methylcytidine which. If deamlnated, produces methylthymidine In DNA. Methylatlon of the bases can modify gene transcription (see text). (PR 5 -phosphor1bose).

Either hydroxyl group of catechol can be methylated by catechol O-methyltransferase, albeit at different rates (/.c., the enzyme does not exhibit absolute regiospecifi-city). The / cat value for the 3-hydroxyl group is about 1 s whereas that at the 4-position is about 0.1 or 0.2 s b The mechanism has been reported to be ordered with SAM binding first, followed by magnesium ion, and then catechol. Interestingly, it appears that the rate-limiting step is the actual catalytic event. 

The methyl transferases (MTs) catalyze the methyl conjugation of a number of small molecules, such as drugs, hormones, and neurotransmitters, but they are also responsible for the methylation of such macromolecules as proteins, RNA, and DNA. A representative reaction of this type is shown in Figure 4.1. Most of the MTs use S-adenosyl-L-methionine (SAM) as the methyl donor, and this compound is now being used as a dietary supplement for the treatment of various conditions. Methylations typically occur at oxygen, nitrogen, or sulfur atoms on a molecule. For example, catechol-O-methyltransferase (COMT) is responsible for the biotransformation of catecholamine neurotransmitters such as dopamine and norepinephrine. A-methylation is a well established pathway for the metabolism of neurotransmitters, such as conversion of norepinephrine to epinephrine and methylation of nicotinamide and histamine. Possibly the most clinically relevant example of MT activity involves 5-methylation by the enzyme thiopurine me thy Itransf erase (TPMT). Patients who are low or lacking in TPMT (i.e., are polymorphic) are at... 

A large number of both endogenous and exogenous compounds can be methylated by several N-, 0-, and S-methyl transferases. The most common methyl donor is S-adenosyl methionine (SAM), which is formed from methionine and ATP. Even though these reactions may involve a decrease in water solubility, they are generally detoxication reactions. Examples of biologic methylation reactions are seen in Figure 7.18. 

Microcontact printing (pCP, see Fig. 10 for an example) has been used for the spatially resolved modification of gold, silver, or titanium surfaces with SAMs of methyl-terminated alkanethiolates, which favor protein adsorption [99-101], Backfilling around the patterned protein-attractive islands was performed by a subsequent self-assembly of an ethylene-glycol-terminated alkanethiol. In a next step, the hydrophobic methyl-terminated SAMs were covered by adsorbed FN or other cell-adhesion-mediating proteins. 

The major (salvage) pathways for the formation of phosphatidylcholine and ethanolamine are illustrated in Figure 19.16. Free (dietary) choline and etha-nolamine are converted to their CDP derivatives, which then react with diacyl-glycerol to form phosphatidylcholine and ethanolamine. In the lungs, another pathway forms dipalmitoyl phosphatidylcholine, a powerful surfactant. Phos-phatidylethanolamine may be methylated by S-adenosylmethionine (SAM see Chapter 20) to yield phosphatidylcholine. The reaction is catalyzed by two enzymes the first methyl group is transferred via phosphatidylethanolamine N-methyltransferase I. The other two methyl groups are transferred by phosphatidylethanolamine N-methyltransferase II. Some authorities believe that the two enzymes are identical. It has also been proposed that methylation of phospha-... 

SAM) by methionine adenosyltransferase. SAM serves as a methyl donor for a variety of methyl acceptors, including DNA, protein, neurotransmit-ters, and phospholipids. 5-Adenosylhomocysteine (SAH) is produced following methyl donation by SAM, and homocysteine is formed through the liberation of adenosine from SAH by the enzyme SAH hydrolase. Unlike methionine and cysteine, homocysteine is not incorporated into polypeptide chains during protein synthesis. Instead, homocysteine has one of two metabolic fates transsulfuration or remethylation to methionine. 

Finally, three methylations on the nitrogen atom by SAM (see p. 1348) gives the zwitterion phosphatidyl choline. 2... 

The cyclization product is still an amino acid and it can be decarboxylated by pyridoxal. Now we have something quite like papaverine but it lacks the methyl groups and the aromatic heterocyclic ring. Methylation needs SAM and is done in two stages for a reason we will discover soon. The final oxidation should again remind you of the closing stages of the tropinone route. 

With the assumption that reticulines are also precursors in mammalian synthesis of morphine, it was challenging to investigate whether they could be produced by enzymatic reactions similar to those utilized in benzylisoquinoline-producing plants (274). This plan focused attention on reactions controlled by the enzyme catechol 0-methyltransferase (COMT), using 5-adenosyl-L-methionine (SAM) for the methylation reaction. Mammalian COMT is present in mammalian tissues, particularly the liver, and an enzyme preparation from rat liver was used for the experiments. It was found that (S)-norcoclaurine, which is the first isoquinoline produced in benzylisoquinoline-producing plants, was similarly O-methylated in vitro by SAM in the presence of COMT, and a reverse proportion of methylated products was obtained with the (/ )-enantiomer (277). Similar 0-methylation of (5)-4 -demethylreticuline (3 -hydroxy-N-methylcoclaurine), prepared by total synthesis (162), however, afforded almost exclusively (5)-orientaline, with a methoxy group at C-3 and not at C-4 as in (5)-reticuline (Fig. 37) (762). 

The catalytic mechanism of Icmt is proposed to be an ordered bi-bi kinetic reaction [38,39]. In this mechanism, the methyl donor, SAM, binds to the active site first, followed by binding of the prenylcysteine substrate. Once the transfer reaction is complete, the methylated product is released followed by dissociation of 5-adenosyl-L-homocysteine (AdoHcy, SAH). Further biochemical and biophysical experiments are underway to further elucidate the mechanism of methyl donor and acceptor binding and catalysis of these unique enzymes. 

COMT (EC 2.1.1.68) was detected as an enzyme methylating caffeic acid to ferulic acid with the help of SAM and later it was shown that 5-hydroxyferulic acid is also methylated to sinapic acid. Mostly, other substrates will be accepted as well (Roje, 2006). Nowadays, however, there are strong indications that the hydroxycinnamic aldehydes and/or alcohols will be preferentially methylated by COMTs (Parvathi et al, 2001). Kinetic investigations of the... 

One of the hydroxyl groups of (S)-norcoclaurine is methylated by a S-adenosyl methionine-(SAM)-dependent O-methyl transferase to yield (S)-coclaurine. This enzyme has been cloned, and the heterologously expressed enzyme exhibited the expected activity (15-17). The resulting intermediate is... 

Cobalamin-dependent methionine synthase contains a built-in repair mechanism. If accidental oxidation of cob(I)alamin leads to inactive cob(lI)alamin, then the enzyme employs SAM and reduced flavodoxin to regenerate cob(I)alamin. Although the redox equilibrium below lies mainly on the left side, any cob(l)alamin formed is trapped by SAM-dependent methylation to yield methylcobalamin. 

The methylation cycle proceeds as follows. Methionine can be converted to S-adenosylmethionine (SAM). SAM is a universal methyl donor and is required in most or alJ methylation events occurring in the body. For example, SAM is used in the synthesis of creatine and carnitine and in the methylation of nucleic acids and proteins. With the donation of the methyl group, SAM is converted to S-ade-nosylhomocystcine (SAH), as shown in Figure 9-4. SAH is finally broken down to homocysteine, completing the methylation cycle. The point of departure of the 1-carbon unit, derived from serine, from the methylation cycle is indicated by the section symbol (g). 

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