Methylation, with SAM

Some features of the biosynthesis of mirabimide E (94) were examined through feeding experiments with [l,2- C2]acetate and [methyl- C]methionine, which showed the decanoate and C-4 and C-5 of the pyrrolinone unit to derive from six units of acetate, and the C- and 0-methyl groups to derive from methionine. Considering the models provided by jamaicamide A (93) ° and barbamide (78), it seems likely that the decanoate may derive from hexanoate, which is multiply halogenated by a radical halogenase related to that described from barbamide. This would be extended by two rounds of PKS extension, C-methylation with SAM and simple ketoreduction of the /3-carbonyl formed after the second extension. An NRPS with specificity for L-alanine is likely responsible for amide bond formation and this product is then extended by one more acetate... 

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, which are introduced by methylation with SAM and oxidation. 

MetH catalyzes the methylation of the bound and reduced cob(I)alamin cofactor by (N -protonated) N -methyltetrahydrofolate to give enzyme-bound methylcobalamin (3) in a base-off/His-on form (see later) [125,153-155]. The methyl-Co(III)corrinoid is demethylated by homocysteine, whose sulfur is activated and deprotonated due to the coordination to a zinc ion (held by three cysteine residues) of the homocysteine binding domain [164] (see Fig. 15). The two methyl-transfer reactions occur in a sequential mechanism [124,125,153,154]. Intermittently, the bound Cob(I)alamin (40 ) is oxidized to enzymatically inactive cob(II)alamin (23) and requires reactivation by reductive methylation with SAM and a flavodoxin as a reducing agent [125,153-155,165]. 

The first biosynthetic studies on azinomycins, which appeared in 2004, reported that sodium acetate labeled at C-1, C-2 or doubly labeled with was each fed to S. sahachiroi at the onset of azinomycin B production. Such experiments revealed that the naphthoate fragment of azinomycin B is of polyketide origin. It was proposed that condensation of one molecule of acetyl-CoA with five of malonyl-CoA leads to linear polyketide 22, catalyzed by a PKS. Further reduction, cyclization, and aromatization of the linear polyketide 22 by the PKS led to the carboxylic acid 23, which would be hydroxylated to give 24, methylated with SAM to give 25 and incorporated into the complete azinomycin B skeleton (Figure 3.10) [19]. 

The biosynthesis of prenylated benzoic acid 37 starts from prephenate, which is converted to 4HPP 38 catalyzed by novF. The prenylation with DMAPP is catalyzed by a prenyltransferase (gene novQ) [164]. Gene novR catalyzes the oxidative transformation to prenylated benzoic acid 37 [157]. The aminocumarin 36 is assembled with 3-prenyl-4-hydroxybenzoate 37 by novL. Subsequent methylation with SAM catalyzed by novO affords the compound 42. Gene novM catalyzes the assembly of the latter with dTDP-L-noviose 32. Finally, a 4"-(9-methyl transfer (gene novP) and a 3"-0 carbamyl transfer (gene novN) form novobiocin [165]. 

Despite the higher selectivity of enzymatic methyl transfer over chemical methylation, where toxic or hazardous reagents are often employed, such as methyl sulfonate and diazomethane, the synthetic applications of these enzymes have been largely ignored primarily as a result of high costs associated with the cofactor SAM. Recent efforts have been directed to in vivo methylation, where SAM may be regenerated inside cells. For example, methyl benzoate production was engineered in recombinant Saccharomyces cerevisiae and in vivo... 

Lysine is the most common site for N-methylation, but methylation can also occur on arginine, histidine, glutamine, and asparagine. The enzymes responsible for N-methylation are known as N-methyltransferases aided with SAM as a cosubstrate. All forms of methylation share the same mechanism the nucleophilic amino acid side chain attacks the electrophilic methyl group of SAM and releases S-adenosylhomocysteine (SAH) (Scheme 7). 

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. 

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 catabolism of lysine merges with that of tryptophan at the level of (3-ketoadipic acid. Both metabolic pathways are identical from this point on and lead to the formation of acetoacetyl-CoA (Figure 20.21). Lysine is thus ketogenic. It does not transaminate in the classic way. Lysine is a precursor of carnitine the initial reaction involves the methylation of e-amino groups of protein-bound lysine with SAM. The N-methylated lysine is then released proteolytically and the reaction sequence to carnitine completed. See Equation (19.6) for the structure of carnitine. 

An external aldimine forms with SAM, which is deprotonated to form the quinonoid intermediate. The deprotonated carbon atom attacks the carbon atom adjacent to the sulfur atom to form the cyclopropane ring and release methyl thio-adenosine, the other product. 

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). 

Methylation is the addition of a carbon atom to a molecule, usually causing a change in the function of the methylated molecule. For example, methylation of the neurotransmitter dopamine by catechol-O-methyltransferase renders it inactive. With only two exceptions, 5-adenosylmethionine (SAM), an activated form of the essential amino acid methionine, is the methyl donor for each of the more than 150 methylation reactions, which regulate a large number of cellular functions. One exception is methylation of homocysteine (HCY) to methionine by the cobalamin (vitamin Bi2)-dependent enzyme methionine synthase, which utilizes 5-methyltetrahydrofolate (methylfolate) as the methyl donor, serving to complete the methionine cycle of methylation, as illustrated in Fig. 1 (lower right). Notably, HCY formation from S-adenosylhomocysteine (SAH) is reversible and, as a result, any decrease in methionine synthase activity will be reflected as an increase in both HCY and SAH. This is significant because SAH interferes with SAM-dependent methylation reactions, and a decrease in methionine synthase activity will decrease all of these reactions. Clearly methionine synthase exerts a powerful influence over cell function via its control over methylation. 

Fusarin C 4 is a mycotoxin produced by a number of Fusarium species. These organisms are often significant pests of cereals. Isotopic labelling studies indicated that it is biosynthesised from a polyketide chain fused to a C4N unit most probably derived from an amino acid. Once again the polyketide moiety is methylated with carbon atoms from SAM (22). 

After serotonin is produced within the pineal gland, it is converted to 5-hydroxy-N-acetyltryptamine by N-acetyl transferase. 5-Hydroxy-N-acetyl-tryptamine is then methylated by O-methyl transferase. SAM is the methylating agent. With this information, draw the synthetic pathway of melatonin. 

The methylation steps alternate with the three hydroxylations described above introducing methyl groups at 6-OH, at the ring C-3, and at the 5-OH group, respectively. The three methyl groups are derived from methionine, with SAM being the actual methyl donor. ... 

Evidence relating to the exact roles of SAM and methylcohalamin in the action of MetH were confusing for many years. It was clear that the generic methyl donor SAM was required for activity, hut methyltetrahydrofolate was the better methyl donor to homocysteine. The first evidence for the participation of cob(I)alamin came with the observation that propyl iodide inactivated the enzyme by alkylation of cob(I)alamin to propylcobalamin. The propylated enzyme was reactivated by light. A confusing aspect was that the propylated enzyme could still catalyze methyl transfer from methylcohalamin to homocysteine. 

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