S-Inosyl-l-Homocysteine Hydrolase, a Novel Enzyme Involved inS-Adenosyl-l-Methionine Recycling

Abstract

S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl-L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD ؉ and has K m values of 0.64 ؎ 0.4 mM, 0.0054 ؎ 0.006 mM, and 0.22 ؎ 0.11 mM for inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. IMPORTANCEIn strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the biochemical reactions that were occurring when life originated. The recycling of S-adenosyl-L-methionine (SAM)-derived metabolites in Methanocaldococcus jannaschii was recently shown to use a novel enzyme, 5=-deoxyadenosine deaminase (DadD) (1). DadD deaminates three SAM-derived enzymatic products (5=-methylthioadenosine, 5=-deoxyadenosine, and S-adenosyl-L-homocysteine) to produce the inosine analogs ( Fig. 1) (1). The canonical pathway for recycling S-adenosyl-L-homocysteine (SAH) produced from SAM-dependent methyltransferases (2-4) proceeds in a three-step recycling pathway back to SAM (Fig. 2A). SAH is first hydrolyzed to produce adenosine and homocysteine using S-adenosyl-L-homocysteine hydrolase (SAHH). Homocysteine is then methylated by methionine synthase to produce methionine (5). The last step in the pathway is the generation of SAM by combining the adenosyl moiety of ATP with methionine to produce SAM, pyrophosphate, and phosphate (Fig. 2), catalyzed by SAM synthase (6). A similar pathway has been established in M. jannaschii but with ...

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A bicyclic S-adenosylmethionine regeneration system applicable with different nucleosides or nucleotides as cofactor building blocks

“…34 Nevertheless, all enzymes have to accept the modified substrates; representatives of each enzyme family are described to accept (co)substrate analogues but mostly with lower conversions (detailed information in Table S2, ESI †). 15,18,19,23,25,27,29,[43][44][45][46][47][48][49][50][51] Besides EcMAT, another MAT originating from Thermococcus kodakarensis, TkMAT, which is likely to have a broader substrate spectrum like other archaeal MATs, was then tested in this setup. 44 Employing TkMAT decreased the MT conversion compared to EcMAT from 43% (22 TTN) to 23% (11 TTN, Table S6, ESI †).…”

“…DNA is comprised of different nucleobases, and the inosine analogue of SAH, S-inosyl-L-homocysteine (SIH, 4c) has been found in the archaeon Methanocaldococcus jannaschii and the bacterium Streptomyces flocculus. 45,64,65 Indeed, SIH seems to be part of an alternative SAM and L-methionine recycling pathway in archaea. 45,66 The experiment using 7-deazaadenosine described above already showed that, in principle, cofactor building blocks with altered nucleobases can be fed in the cycle.…”

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