S-adenosylhomocysteine metabolism in Streptomyces flocculus

Abstract

S-Adenosylhomocysteine metabolism was studied in cell extracts of streptonigrin-producing Streptomyces flocculus. The major route of metabolism was found to be deamination to form S-inosylhomocysteine. The metabolite was purified by high-performance liquid chromatography and identified by its UV and nuclear magnetic resonance spectra and by its chemical degradation to hypoxanthine.Streptomyces flocculus (ATCC 13257) produces the antitumor antibiotic streptonigrin (9). The biosynthetic pathway which yields streptonigrin involves an S-adenosylmethionine-mediated C-methylation of the I position of the side chain of tryptophan to form P-methyltryptophan (7), as well as two 0-methylations later in the pathway (6). The tryptophan C-methyltransferase is the first enzyme in the pathway which commits a primary metabolite to antibiotic biosynthesis and therefore is a likely site of regulation for the pathway. One product of the reaction, S-adenosyl-L-homocysteine (AdoHcy), is known to be a potent inhibitor of S-adenosylmethionine-dependent methyltransferases (2, 4), and inhibition of the tryptophan C-methyltransferase by AdoHcy has been demonstrated in partially purified enzyme preparations (M. K. Speedie and B. Fox, unpublished data).Cellular levels of AdoHcy are known to be regulated in eucaryotic cells by AdoHcy hydrolase (EC 3.3.1.1), which hydrolyzes AdoHcy to adenosine and homocysteine (12). In procaryotic organisms, the predominant route of metabolism is catalyzed by AdoHcy nucleosidase (EC 3.2.2.9), which generates adenine and S-ribosylhomocysteine (14), although recently Shimizu et al. (12) demonstrated the occurrence of AdoHcy hydrolase in some procaryotic organisms, including one streptomycete. In order to examine AdoHcy metabolism in S. flocculus, we incubated cell extract with AdoHcy and discovered an unexpected metabolite, S-inosylhomocysteine (InoHcy), as the major product. The identification of this metabolite is the subject of this report.Cultures of S. flocculus (ATCC 13257) were inoculated with a 5% inoculum of seed culture which had been grown for 48 h from a spore suspension in 100 ml of Emerson broth (1.0% glucose, 0.4% beef extract, 0.4% Gelysate peptone, 0.1% yeast extract, 0.25% NaCl in 1 liter of distilled water, adjusted to pH 7.0) in a 1-liter Erlenmeyer flask at 28°C and 180 rpm. Cultures (250 ml of Emerson broth in 2-liter Erlenmeyer flasks) were grown for 24 h at 28°C and 180 rpm (New Brunswick model M52 Controlled Environment Incubator Shaker) and then harvested by filtration. A cell slurry in 10 mM sodium phosphate buffer (pH 8.0) with 1 mM dithiothreitol was sonified at setting 5 on an Ultrasonics sonifier in an ice bath and centrifuged for 20 min at 30,000 x g to remove cell debris. The crude cell extract was precipitated with 70% ammonium sulfate and centrifuged at 30,000 x g for 20 min at 6°C. The precipitate was redissolved in 20 mM potassium phosphate buffer, pH 7.0, and dialyzed for 24 * Corresponding author. h against two changes (2.5 liters) of the same buffer at 4°C. This prepar...

Identification of a New Class of Adenosine Deaminase from Helicobacter pylori with Homologs among Diverse Taxa

“…Hp0267 acts on both adenosine and SAH, with k cat /K m values that suggest that either of these reactions could be the enzyme's primary physiological role. Nevertheless, if SAH were indeed a major substrate for Hp0267 or for its ortholog Tm0930, this would imply that a degradation pathway is present for SAH, of which only one is known to exist in bacteria (34). Our comparative genomic data reveal that the presence of an Hp0267 homolog correlates with the absence of a cog1816 ADD.…”

“…This high affinity for SAH was nevertheless tempered by a 30-fold-lower V max compared with that of adenosine as a substrate. One explanation for the observed activity on SAH could be that the physiological role for Hp0267 is as an SAH deaminase; however, there is only one such enzyme described in the literature (from Streptomyces flocculus), and the specifics of this degradation pathway are uncertain (33,34). In addition, it was shown that Tm0936 catalyzes the deamination of adenosine nearly as efficiently as for SAH, and furthermore, the T. maritima genome lacks a homolog to adenosine deaminase (25).…”

Identification of a 5'-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5'-Deoxyadenosine

“…At this time only the enzymes from Thermotoga maritima (1), Pseudomonas aeruginosa (3), and Streptomyces flocculus (17) have been studied. None of these enzymes were ever tested with 5=-deoxyadenosine (5=-dA) as the substrate.…”

Purification and characterization of S-adenosylhomocysteine deaminase from streptonigrin-producing Streptomyces flocculus

“…In summary, AdoHcy is metabolized to InoHcy in S. flocculus by a deaminase which represents the major route for AdoHcy in this organism (15). Because AdoHcy is a potent inhibitor of S-adenosylmethionine-dependent transmethylases, its efficient removal by the organism can play an important role in controlling methylation reactions.…”

“…When the cell extracts from S. flocculus were examined for AdoHcy nucleosidase and hydrolase activities, neither of these activities was observed, but an unknown metabolite, S-inosylhomocysteine (InoHcy), was determined to be the major product (15). Because of its potential regulatory role and its novelty with regard to AdoHcy metabolism, the deaminating enzyme responsible for the conversion of AdoHcy to InoHcy was purified and characterized.…”