SAM synthetase

Scheme 1. The reactions of the Streptomyces cattleya cell-free extract incubation of ATP 7 and fluoride ion, illustrating the formation of 5 -FDA 5, 5 -FDI 6 and FAc 1. L-methionine is recycled between SAM synthetase and the fluorinase [7].

Some of the enzymes involved in this reaction pathway have been identified. The enzyme of the first step in the reaction pathway, SAM synthetase, is a known enzyme in plant tissues (15). The enzyme converting SAM to ACC has been isolated from tomato tissues (14) and appears to be a pyridoxal phosphate-mediated enzyme. However, the enzyme converting ACC to ethylene has not been isolated as yet, although indications are that it reacts with oxygen by a complex mechanism, perhaps to form free-radical intermediates. 

In a classic paper, Snapper synthesized an ilimaquinone-agarose-affinity resin (30), which was incubated with homogenized bovine liver and then washed extensively.93 Proteins retained by the resin were separated by gel electrophoresis, yielding six main protein bands. Amino acid sequencing of these bands revealed three proteins involved in the activated methyl cycle — SAHase, S-adenosylmethionine synthetase (SAM synthetase), and catechol-O-methyltransferase (COMT) — as well as three unrelated proteins. Subsequent enzymatic assays established that ilimaquinone is a competitive inhibitor of SAHase, but has little effect on the activity of SAM synthetase or COMT. The authors noted that a consequence of SAHase inhibition would be the intracellular accumulation of SAH, which is a potent feedback inhibitor of methyltransferases. These results support the assertion that methylation events play an important role in cellular secretory events and vesicle-mediated processes. The study also highlighted the problem of nonspecific interactions as only one of the six isolated proteins was shown to interact in any way with the natural product. 

The current status of the overexpression and purification of SAM synthetase is summarized in the table. 

The SAM synthetase is highly regulated, as an overproduction of SAM would have dramatic impact on many cellular SAM-dependent pathways. Nevertheless, some SAM overproducers have been constructed in the recent years [33]. Access to cheap SAM or an efficient recycling system is the prerequisite for applications of SAM-dependent MTs in biocatalysis and production of methylated small molecules as active pharmaceutical ingredients (APIs) or mediators to APIs and even high-value specialty chemicals (for more details, see Section 18.2.4). 

With purified SAM synthetase, SAM can be synthesized from L-Met 7 and ATP. The enzymes from E. coli, yeast, and rat liver have high substrate selectivity, and only SAM 1 can be synthesized efficiently by applying the enzymatic approach [34]. 

Floss and coworkers [35] analyzed the stereochemistry of the MT-catalyzed transmethylations. For this purpose, non-racemic [methyl- Hj, Hj]SAM was synthesized, starting from [methyl- Hj, Hj]methionine and ATP, catalyzed by SAM synthetase. The MT and substrates were incubated, the methylated products were converted to [methyl- Hj, Hj]acetic add, and the chirality was determined by the method of Arigoni and Cornforth [36]. These investigations led to the confirmation of the Sj 2-type reaction with inversion of the configuration (Scheme 18.6) [37]. 

MAT was SAM synthetase 11 from Saccharomyces cerevisiae unless otherwise stated... 

The SAM synthetase (different from the Met-transfer RNA synthetase) although discriminative against ethionine, nevertheless utilizes it. Subsequent enzymes then catalyse the transfer of the ethyl group which has been found in nucleic acids from rat liver. The exact biological significance of such replacements is not known. 

Fig. 5. Regulation of the enzymes of methionine biosynthesis and related pathways. Enzymes catalyzing the synthesis of methionine and 5 -adenosylmethionine (SAM) from cysteine are (1) cystathionine y-synthase, (2) j9-cystathionase, (3) methionine synthase, and (4) SAM synthetase. Enzymes associated with the synthesis and metabolism of phospbohomoserine which are relevant to the regulation of methionine synthesis are (5) aspartate kinase, (6) homoserine kinase, and (7)...

Fig, 7. Pathways for the metabolism of methionine to 5 -methylthioadenosine (MTA) and recycling of MTA to methionine. Methionine can serve as a carbon source for the synthesis of polyamines and, in some tissues, ethylene. 5 -Methylthioadenosine is a product of both processes. Only the methylthio group of methionine is recycled, the C4 moiety for the resynthesis of methionine being derived from the ribosyl moiety of ATP. The enzymes involved are (1) SAM synthetase, (2) SAM decarboxylase, (3) various C3 transfer enzymes of polyamine biosynthesis, (4) MTA nucleosidase, (5) methylthioribose kinase, (6) three( ) uncharacterized enzymes, (7) aminotransferase, and (8) aminocyciopropane carboxylate synthase. 

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