Escherichia coli S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action

Abstract

S-Adenosylhomocysteine/5'-methylthioadenosine nucleosidase (EC 3.2.2.9) was purified to homogeneity from Escherichia coli to a final specific activity of 373 ,umol of 5'-methylthioadenosine cleaved/min per mg of protein. Affinity chromatography on S-formycinylhomocysteine-Sepharose is the key step of the purification procedure. The enzyme, responsible for the cleavage of the glycosidic bond of both Sadenosylhomocysteine and 5'-methylthioadenosine, was partially characterized. The apparent Km for 5'-methylthioadenosine is 0.4 /LM, and that for S-adenosylhomocysteine is 4.3 /tM. The maximal rate of cleavage of S-adenosylhomocysteine is approx. 40% of that of 5'-methylthioadenosine. Some 25 analogues of the two naturally occurring thioethers were studied as potential substrates or inhibitors of the enzyme. Except for the analogues modified in the 5'-position of the ribose moiety or the 2-position of the purine ring, none of the compounds tested was effective as a substrate. Moreover, 5'-methylthioformycin, 5'-chloroformycin, S-formycinylhomocysteine, 5'-methylthiotubercidin and S-tubercidinylhomocysteine were powerful inhibitors of the enzyme activity. The results obtained allow the hypothesis of a mechanism of enzymic catalysis requiring as a key step the protonation of N-7 of the purine ring.

Towards area‐based in vitro metabolic engineering: Assembly of Pfs enzyme onto patterned microfabricated chips

“…Reported values of the Michaelis-Menten constant K m have varied widely. 21,29 Duerre 21 reports a K m of 3 mM, while Ragione et al 29 report a K m of 4.3 lM. This discrepancy in K m values is attributed by Ragione et al to differences in enzyme purity and in the assay used to determine conversion; we also have used different purification and analytical techniques.…”

“…27 For this, several researchers have designed inhibitors of Pfs activity as possible antibiotics, and such research is ongoing. [28][29][30][31] Thus, assembling Pfs for the controlled examination of its catalytic activity could be used to screen Pfs inhibitors with antibiotic capabilities.…”

Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports That Menaquinone Synthesis Occurs through the Futalosine Pathway

“…MTANs are required in a diverse array of biosynthetic pathways (37)(38)(39) that have yet to be implicated in Chlamydia (e.g. polyamine biosynthesis, quorum sensing, DNA methylation, etc.).…”

“…6A), the enzymatic characterization of CT263 indicates that only AFL can be hydrolyzed at a physiological rate. Traditionally, MTANs hydrolyze intermediates of AdoMet utilization pathways (37)(38)(39), which include substrates that were not processed by CT263 (e.g. MTA and AdoHcy, Fig.…”

“…Identification of CT263 Substrates and Hydrolyzed Products-MTAN enzymes have been demonstrated to hydrolyze a variety of substrates, including 5Ј-methylthioadenosine (MTA), AdoHcy, and 6-amino-6-deoxyfutalosine (AFL) (37)(38)(39). Typically, these enzymes catalyze N-glycosidic bond hydrolysis between N9 of adenine and C1 of the thioribose.…”